Lymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereof

ABSTRACT

This invention relates to lymphotoxin-β, a lymphocyte membrane type protein. This protein is found on the surface of a number of cells, including phorbol ester (PMA) stimulated T cell hybridoma II-23.D7 cells. This invention also relates to complexes formed between lymphotoxin-β and other peptides such as lymphotoxin-α and to complexes comprising multiple subunits of lymphotoxin-β. These proteins and complexes are useful in holding LT-α formed within the cell on the cell surface where the LT-α/LT-β complex may act as an inflammation regulating agent, a tumor growth inhibiting agent, a T cell inhibiting agent, a T cell activating agent, an autoimmune disease regulating agent, or an HIV inhibiting agent. Furthermore, the antitumor activity of the LT-α/LT-β complex may be delivered to tumor cells by tumor infiltrating lymphocytes (TILs) transfected with the gene for LT-β.

[0001] The present application is a continuation-in-part of co-pendingapplications PCT/US91/04588, filed Jun. 27, 1991 and U.S. PatentApplication. Ser. No. 07/544,862, filed Jun. 27, 1990.

[0002] The invention described herein was made in part during the courseof work under Grant No. CA 35638-07-10 from the National Institutes ofHealth. The U.S. Government has certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

[0003] This invention relates to lymphotoxin-β, a lymphocytemembrane-type polypeptide. Lymphotoxin-β, also referred to as p33, hasbeen identified on the surface of T lymphocytes, T cell lines, B celllines and lymphokine-activated killer cells.

[0004] This invention also relates to complexes formed betweenlymphotoxin-β (LT-β) and other lymphotoxin-type peptides such aslymphotoxin (which we refer to herein as “lymphotoxin-α (LT-α)” todistinguish it from LT-β) and to complexes comprising multiple subunitsof LT-β. The LT-β polypeptide of this invention is expected to be usefulin holding LT-α formed within the cell on the cell surface where eitherLT-β or the LT-α/LT-β complex may act as an inflammation regulatingagent, a tumor growth inhibiting agent, a T cell inhibiting agent, a Tcell activating agent, an immunomodulatory agent, an autoimmune diseaseregulating agent or an HIV regulating agent. Furthermore, the antitumoractivity of the LT-α/LT-β complex may be delivered to tumor cells bytumor infiltrating lymphocytes (TILs) transfected with the gene forLT-β.

BACKGROUND OF THE INVENTION

[0005] The initiation of the immune response involves a complex array ofintercellular signals. These signals typically involve soluble cytokinescoupled with a number of cell-cell contact dependent signals. Thecontact dependent events, most notably activation of the T-cellreceptor, lend specificity to the response whereas the soluble mediatorsare generally responsible for maintenance of cell differentiation andproliferation. Tumor Necrosis Factor (TNF) and LT-α are two polypeptidesgenerally recognized for involvement with the initiation of the immuneresponse.

[0006] TNF and LT-α are soluble proteins noted originally for theirability to inhibit the growth of tumors. [L. Old, “Tumor NecrosisFactor,” Science, 230:630 (1985)]. Further research demonstrated thatboth proteins exhibit a wide range of activities. TNF is synthesized inresponse to various inflammatory insults by a variety of cell typesincluding both hematopoietic and nonhematopoietic cells, while LT-α, incontrast, is made specifically by lymphocytes. The two known TNFreceptors do not appear to discriminate between LT-α and TNF. [T. Schallet al., “Molecular Cloning and Expression of a Receptor for Human TumorNecrosis Factor,” Cell, 61:361-370 (1990); C. Smith et al., “A Receptorfor Tumor Necrosis Factor Defines an Unusual Family of Cellular andViral Proteins,” Science 248:1019 (1990)]. In general, LT-α and TNFdisplay similar spectra of activities in in vitro systems, although LT-αis often less potent. [J. Browning et al., Studies Of The DifferingAffects Of Tumor Necrosis Factor And Lymphotoxin On The Growth OfSeveral Human Tumor Lines,” J. Immunol., 143:1859 (1989)].

[0007] TNF appears to play a major role in specific aspects of metaboliccontrol, in the response to endotoxin shock, and in the regulation ofhematopoietic cell development. [B. Beutler et al., “The History,Properties, and Biological Effects of Cachectin,” Biochemistry, 27,(1988); M. Akashi et al., “Lymphotoxin: Stimulation And Regulation ofColony Stimulating Factors in Fibroblasts,” Blood, 74:2383 (1989); G.Roodman et al., “Tumor Necrosis Factor-alpha and HematopoieticProgenitors: Effects Of Tumor Necrosis Factor On The Growth Of ErythroidProgenitors CFU-E And BFU-E And The Hematopoietic Cell Lines k562, HL60,And HEL Cells,” Exp. Hematol., 15:928 (1987)].

[0008] Along with IL-1 and IL-6, TNF is also a major mediator of theinflammatory response. [D. Cavender et al., “Endothelial Cell ActivationInduced By Tumor Necrosis Factor And Lymphotoxin,” Amer. Jour. Path.,134:551 (1989); R. Cotran et al., “Endothelial Activation Its Role InInflammatory And Immune Reactions,” in Endothelial Cell Biology, (PlenumPress, Simonescu & Simonescu, eds., 1988) 335]. TNF also appears to beinvolved in T cell activation under certain conditions. [M. Shalaby etal., “The Involvement Of Human Tumor Necrosis Factors-α And-β In TheMixed Lymphocyte Reaction,” J. Immunol., 141:499 (1988); N. Damle etal., “Distinct Regulatory Effects of IL-4 and TNF-α During CD3-Dependentand CD3-Independent Initiation Of Human T-Cell Activation,” Lymph. Res.,8:85 (1989); G. Ranges et al., “Tumor Necrosis Factor-a As AProliferative Signal For An IL-2-Dependent T Cell Line: Strict SpeciesSpecificity of Action,” Amer. Assoc. Immunol., 142:1203 (1989); G.Ranges et al., “Tumor Necrosis Factor a/Cachectin Is A Growth Factor ForThymocytes,” J. Exp. Med., 167:1472 (1988); P. Scheurich et al.,“Immunoregulatory Activity Of Recombinant Human Tumor Necrosis Factor(TNF)-α: Induction Of TNF Receptors On Human T Cells And TNF-α-MediatedEnhancement Of T Cell Responses,” J. Immunol., 138:1786 (1987)].

[0009] TNF is produced by several types of cells, including monocytes,fibroblasts, T cells and Natural Killer (NK) cells. [D. Goeddel et al.,“Tumor Necrosis Factors: Gene Structure And Biological Activities,” ColdSpring Harbor Symposium Quant. Biol., 51, 597 (1986); D. Spriggs et al.,“Tumor Necrosis Factor Expression In Human Epithelial Tumor Cell Lines,”J. Clin. Invest., 81:455 (1988); M. Turner et al., “Human T cells FromAutoimmune and Normal Individuals Can Produce Tumor Necrosis Factor,”Eur. J. Immunol., 17:1807 (1987)]. Investigators have also detectedmurine and human forms of TNF that are associated with the surface ofvarious cells either as a transmembrane protein or a receptor-boundmolecule. [B. Luettig et al., “Evidence For the Existence of Two Formsof Membrane Tumor Necrosis Factor: An Integral Protein and a MoleculeAttached To Its Receptor,” J. Immunol., 143:4034 (1989); M. Kriegler etal., “A Novel Form of TNF/Cachectin Is a Cell Surface CytotoxicTransmembrane Protein: Ramifications For the Complex Physiology of TNF,”Cell, 53, pp. 45-53 (1988); and M. Kinkhabwala et al., “A Novel AdditionTo the T Cell Repertory,” J. Exp. Med., 171:941-946 (1990)].

[0010] LT-α also has many activities, generally similar, but notidentical to those of TNF, including tumor necrosis, induction of anantiviral state, activation of polymorphonuclear leukocytes, inductionof class I major histocompatibility complex antigens on endothelialcells, induction of adhesion molecules on endothelium and growth hormonestimulation. [N. Ruddle and R. Homer, “The Role of Lymphotoxin inInflammation,” Prog. Allergy, 40:162-182 (1988)]. Both LT-α and TNF areligands to members of the nerve growth factor (NGF) receptor family. [S.Mallett and A. N. Barclay, “A New Superfamily Of Cell Surface ProteinsRelated To The Nerve Growth Factor Receptor,” Immunology Today,12:7:220-223 (1991).]

[0011] In contrast to TNF, LT-α secretion appears to be a specificproperty of only activated T cells and certain B-lymphoblastoid tumors.[N. Paul et al., “Lymphotoxin,” Ann. Rev. Immunol., 6:407 (1988)]. Someresearchers have also indicated that a membrane-associated form of LT-αmay be expressed on the surface of lymphocytes under certaincircumstances [J. Hiserodt, et al., “Identification ofMembrane-Associated Lymphotoxin (LT) On Mitogen-Activated HumanLymphocytes Using Heterologous Anti-LT Antisera In Vitro,” Cell.Immunol., 34:326-339 (1977); C. Ware et al., “Mechanisms ofLymphocyte-Mediated Cytotoxicity,” J. Immunol., 126:1927-1933 (1981); U.Anderson et al. J. Immunol. Methods, 123, 233 (1989); Y. Abe et al.,Jpn. J. Canc. Res., 82:23 (1991); Y. Abe et al., “Studies of MembraneAssociated and Soluble (Secreted) Lymphotoxin In HumanLymphokine-Activated T-Killer Cells In Vitro,” Lymphokine and CytokineResearch, 11, 2:15-121 (1992)].

[0012] In recent years genes for both TNF and LT-α have been isolatedand cloned, leading to their complete characterization and to theavailability of recombinant forms of both proteins. [P. Gray et al.,“Cloning and Expression of cDNA For Human Lymphotoxin, A Lymphokine WithTumor Necrosis Activity,” Nature, 312:121-124 (1984); D. Pennica et al.,“Human Tumor Necrosis Factor: Precusor Structure, Expression AndHomology To Lymphotoxin,” Nature, 312:724 (1984)].

[0013] Other “cytokine-like” cell surface proteins including the CD40protein have recently been shown to share certain similarities with TNFand LT-α. Like TNF and LT-α, the CD40 protein is a ligand to members ofthe TNF/nerve growth factor (NGF) receptor family. [S. Mallett and N.Barclay, Immunology Today, 12:220-223 (1991)]. The CD40 protein is a277-amino acid protein expressed on the surface of B lymphocytes,epithelial cells, and some carcinoma cell lines. [R. Armitage et al.,Nature, 357:80-82 (1992); T. Farrah and C. Smith, “Emerging CytokineFamily,” Nature, 358:26 (1992)].

[0014] We have now identified a novel surface protein, lymphotoxin-β(LT-β) or p33. LT-β has been identified on the surface of several typesof lymphocyte cells, including OKT3-stimulated primary T cells, antigenspecific IL-2 dependent CTL clones, and a PMA-stimulated human T cellhybridoma II-23.D7. LT-β targets LT-α produced in the cell to the cellmembrane where LT-β and LT-α appear as a complex (designated “LT-α/LT-β”throughout this disclosure). The LT-α/LT-β complex is believed to be anovel mechanism for membrane expression of LT-α by activated T-cells.[Androlewicz et al., “Lymphotoxin Is Expressed As a Heteromeric complexWith A Distinct 33 kDa Glycoprotein On The Surface Of An Activated HumanT Cell Hybridoma,” Journal Of Biological Chemistry, 267:2542-2547(1992)]. The LT-α/LT-β complex may exhibit cytolytic and cell regulatoryactivity similar to the soluble LT-α, TNF and CD40 proteins. Themembrane-associated LT-β complexed with LT-α may represent, as acomplex, a novel ligand for T cell interactions with other cells and mayalso be useful in targeted cell lysis.

SUMMARY OF THE INVENTION

[0015] The novel protein of the present invention has been namedlymphotoxin-β (LT-β). This protein is found on the surface of severaltypes of lymphocyte cells, including OKT3-stimulated primary T cells,antigen-specific IL-2 dependent CTL clones, and a PMA-stimulated human Tcell hybridoma, II-23.D7. It forms a novel complex with LT-α and formscomplexes with other LT-β subunits (e.g., (LT-β)₂ LT-α complexes).

[0016] LT-β has a molecular weight of 31-35 kD as determined byimmunoprecipitation and SDS-PAGE. LT-β exhibits N-linked glycosylation.The amino acid sequence of lymphotoxin-β is set forth in SEQ ID NO:2,and the amino acid sequences of several soluble lymphotoxin-β peptidesare set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. The DNAsequence coding for lymphotoxin-β is set forth in SEQ ID NO: 1 and DNAsequences coding for several soluble lymphotoxin-β peptides are setforth in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0017] LT-β as a cell membrane protein binds LT-α during synthesis, thus“targeting” the LT-α to the cell membrane. In the absence of LT-β, LT-αis secreted into the extracellular medium. The LT-α/LT-β complex isrecognized by polyclonal antisera raised against recombinantlymphotoxin-α (rLT-α) expressed in CHO cells or by monoclonal antibodies(mAbs) raised against natural LT-α. Furthermore, antisera that recognizethe LT-α/LT-β complex and (LT-α)₃ block the mixed lymphocyte reaction(MLR), a standard immunological assay of the expected proliferativeresponse of T lymphocytes to allogenic stimulation, i.e., theintroduction of T lymphocytes from another individual, which arerecognized as foreign (non-self) by the “responder” lymphocytes. [See,e.g., M. Shalaby et al., J. Immunol., 141:499 (1988)].

[0018] The LT-β protein was purified by affinity chromatography,partially sequenced and a specific oligonucleotide probe was designed.The cDNA encoding the LT-β was isolated by probing a cDNA library fromactivated II-23.D7 cells, a human T-cell hybridoma that displays largeamounts of surface lymphotoxin upon phorbol ester activation. Theidentified amino acid sequence encodes a 240-244 amino acid sequence (amolecular mass of the unmodified protein of about 25130-25390 kDa). SeeSEQ ID NO:2. The amino acid sequence and the placement of thetransmembrane region are typical of a type II membrane protein.

[0019] This sequence comprises a short 14-18 amino acid N-terminal“cytoplasmic” domain. Following this cytoplasmic domain there is anextensive stretch of 30 hydrophobic amino acids which presumably acts asa membrane anchoring domain. No identical sequences were found withinavailable databases. There is one cysteine residue in the extracellulardomain and two methionines within the last C-terminal 17 amino acids.This is consistent with the very limited cyanogen bromide cleavagepattern exhibited by this protein.

[0020] Comparison of the LT-β sequence with other proteins known to bindto members of the TNF/NGF receptor family reveals considerablestructural similarity. Four of the ligands to members of the TNF/NGFreceptor family (TNF, LT-α, LT-β and the CD40 ligand) resemble type IImembrane proteins and share at least four regions of sequenceconservation in the extracellular domain as indicated in FIG. 14. Theconserved TNF and LT-α domains shared with LT-β are likely to beinvolved in intersubunit interactions and B sheet organization. Theseregions of conservation can account for the association between LT-α andLT-β. The existence of these homology regions may facilitate engineeringthe polypeptide to form complexes with, for example, TNF or the CD40ligand. Such a molecule would have mixed functions and could possibly beused as a custom designed drug. [See J. Fuh et al., “Rational Design OfPotent Antagonists To The Human Growth Hormone Receptor,” Science,256:1677 (1992)].

[0021] We believe that the polypeptide complexes of this invention areimportant in T cell activation events and are useful in compositions andmethods for T cell activation or T cell suppression and as therapeuticagents in the treatment of inflammation or applications requiringcytolytic activities, such as inhibition of tumor cell or neoplasiagrowth. We also believe that the polypeptide complexes may be importantin cellular immunotherapies, including enhancing the tumoricidalproperties of tumor infiltrating lymphocytes in Tumor InfiltratingLymphocyte (“TIL”) therapy. TIL immunotherapy may be improved by genetransfer techniques. For example, a gene may be added to tumor cells forthe purpose of inducing the body's immune system to mediate an effectivetumor-directed immune response. [See, e.g., W. F. Anderson, “Human GeneTherapy.” Science, 256:808-813 (1992)].

[0022] We also believe, based upon similarities between a moleculeidentified as “Fas” and members of the TNF/NGF receptor family, that thepolypeptide complexes of this invention may be involved in the internalcell process known as programmed cell death or apoptosis, and maytherefore be involved in mediating autoimmune disease. [See, e.g., N.Itoh et al., “The Polypeptide Encoded By The cDNA For Human Cell SurfaceAntigen Fas Can Mediate Apoptosis,” Cell, 66:233-243 (1991); R.Watanabe-Fukunaga et al., “Lymphoproliferation Disorder In MiceExplained By Defects In Fas Antigen That Mediates Apoptosis,” Nature356:314 (1992)].

[0023] Antibodies to LT-β, its related polypeptides, the LT-α/LT-βcomplex or the other polypeptide complexes of this invention may alsodisrupt critical LT interactions with particular receptors, thusspecifically affecting LT-mediated events other than those mediatedthrough the known TNF receptor forms. Likewise, receptors for TNF, LT-αor LT-α/LT-13, or their derivatives (e.g., soluble receptors andIgG/receptor fusion proteins) may be used to inhibit the polypeptidesand complexes of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 depicts flow cytofluorometric analysis of OKT3-stimulated,IL-2 expanded peripheral blood lymphocytes (PBL) showing reaction with 3different rabbit anti-rLT-α antisera and showing essentially no reactionwith rabbit anti-rTNF antisera.

[0025]FIG. 2 depicts flow cytofluorometric analysis of a human T cellhybridoma, II-23.D7, showing the presence (following PMA treatment) of aLT-α-related epitope on the cell surface.

[0026]FIG. 3 depicts the ability of PMA activated II-23.D7 cells to bindanti-rLT-α antibodies. Samples of anti-rLT-α antisera were incubatedwith PMA-treated U937 (non-LT-producing) cells, (-O-), PMA-activatedII-23.D7 hybridoma cells (10⁸, - - ; 10⁷, -•- ), and no cells(control, - - ). Serial dilutions of the cell-free antisera afterincubation were added to rLT-α and used in a cytotoxicity assay againstL929 (LT-α-sensitive) cells. The plots indicate that rLT-α neutralizingantibodies were removed from the antisera by the activated II-23.D7cells.

[0027]FIG. 4 shows two autoradiographs (A and B) depictingimmunoprecipitation of ¹²⁵I-labeled surface proteins from PMA-activatedII-23.D7 cells. FIG. 4A shows immunoprecipitation of an approximately 25kD surface protein (LT-α) and an approximately 33 kD surface protein(p33, or LT-β) by post-immune rabbit anti-rLT-α antiserum but not bypre-immune rabbit serum. FIG. 4B shows a 1-dimensional CNBr cleavage mapof the 25 kD and 33 kD bands from FIG. 4A, compared against recombinantTNF (rTNF) produced in E. coli, and recombinant lymphotoxin-α (rLT-α)produced in CHO cells, [J. Browning et al., J. Immunol., 143:1859(1989)] both with (+) and without (−) CNBr cleavage.

[0028]FIG. 5 presents autoradiographs showing immunoprecipitation ofTNF- and LT-α-related proteins from PMA-stimulated II-23.D7 cellsmetabolically labeled with ³⁵S-methionine or ³⁵S-cysteine. The figureshows recognition by anti-rLT-α antisera (L), but not preimmune (P) oranti-rTNF antisera (T), of an approximately 25 kD methionine-containingsurface protein (LT-α) and an approximately 33 kD methionine- andcysteine-containing surface protein (p33, or LT-β). The autoradiographsalso indicate that activated II-23.D7 cells also produce a 26 kD form ofTNF and secrete soluble lymphotoxin-α.

[0029]FIG. 6 depicts affinity purification 1-D CNBr peptide mapping ofthose proteins from PMA-activated II-23.D7 cells recognized byanti-rLT-α serum. FIG. 6A represents SDS PAGE analysis of the proteinseluted from an anti-LT-α affinity column prepared from either pre-immune(PRE) or post-immune (POST) rabbit sera. FIG. 6A shows the ˜33 kD and˜20 kD protein bands did not bind to an affinity column prepared usingpreimmune serum (PRE) but did bind to an affinity column prepared usinganti-rLT-α antiserum (POST). FIG. 6B shows partial CNBr cleavage of the⁻33 kD and ⁻20 kD proteins eluted from the POST column, compared againstrTNF and rLT-α run in parallel. The gels were visualized by silverstaining.

[0030]FIG. 7 presents autoradiographs of the ⁻25 kD and ⁻33 kD¹²⁵I-labeled proteins (designated LT-α and LT-B, respectively)immunoprecipitated from activated II-23.D7 cells, treated withN-glycanase (N-gly), with a mixture of neuraminidase and O-glycanase(O-gly), or with all three enzymes.

[0031]FIG. 8 depicts the results of a reimmunoprecipitation of thecoprecipitated p33 (LT-β) and p25 (LT-α) proteins to further investigatewhether they are immunogenically related.

[0032]FIG. 9 (comprising parts 9A and 9B) shows the results ofisoelectric focusing under denaturing conditions of theimmunoprecipitated p33 (LT-β) and p25 (LT-α) proteins.

[0033]FIG. 10 (comprising parts 10A and 10B) shows the results ofisoelectric focusing under native conditions of the immunoprecipitatedp33 (LT-β) and p25 (LT-α) proteins. Together FIGS. 9 and 10 indicatethat LT-β and LT-α form a denaturable complex.

[0034]FIG. 11 depicts flow cytofluorometric analysis of surface proteinsdifferentially expressed on T cells and monocytes after stimulation witha mixture of LPS, IFN-γ and OKT3. From a stimulated PBL pool, separatedT cells were observed to express a surface protein recognized byanti-rLT-α antisera (LT), whereas separated monocytes expressed asurface protein recognized by anti-rTNF antisera (TNF).

[0035]FIG. 12 shows flow cytofluorometric analysis of surface LT formson leu-19⁻ and leu-19⁺ (i.e., natural killer) cells treated with IL-2.Analysis of IL-2 treated PBL with both labeled leu-19 and anti-rLT-αconfirms that lymphokine-activated killer (LAK) cells express a surfaceLT form.

[0036]FIG. 13 depicts amino acid sequence of LT-β fragments obtained bydirect N-terminal sequencing and in situ trypsin digestion followed byreverse phase HPLC resolution of the digested peptides.

[0037]FIG. 14 depicts an amino acid sequence comparison of four membersof the family of ligands binding to members of the TNF/NGF-receptorfamily. Homology regions are shown in bold type face with sequenceidentity indicated with a dot and conserved sequences with an asterisk.Putative N-linked glycosylation sites are boxed.

[0038]FIG. 15 depicts northern analysis of LT-α and LT-β expression. A)Northern blot of several cell lines showing specific expression of bothLT genes in only Hut-78 and II-23.D7 cells. B) Time course of PMAinduction of LT-α and LT-β mRNAs in II-23.D7 cells. C) Similar analysisof human peripheral blood lymphocytes activated with either anti-CD3 orIL-2 alone.

[0039]FIG. 16 depicts expression of LT-α and LT-β in CHO cells. A) FACSanalysis of CHO cells transiently transfected with the LT-β cDNA. Eitherparental dHFR-CHO or LT-α expressing CHO cells were electroporated witheither pCDM8 containing an irrelevant insert (clone 4) or the pCDM8/LT-βplasmid and stained with control IgG( - - - ) or anti LT-α monoclonalantibodies (______). B) Panel B depicts expression of LT-α and LT-β inCOS cells. Cells were transfected with control DNA or the pCDM8/LTβplasmid, either with or without pCDM8/LTα and stained as per panel A.

DETAILED DESCRIPTION OF THE INVENTION

[0040] In order that the invention herein described may be fullyunderstood, the following detailed description is set forth.

[0041] This invention relates to lymphotoxin-B, a lymphocytemembrane-type polypeptide. The amino acid sequence of lymphotoxin-β isset forth in SEQ ID NO:2.

[0042] This polypeptide, also referred to as p33, has a molecular weightof 31 to 35 kD. The polypeptides of this invention may be associatedwith a cell surface or not associated with such a surface.

[0043] This invention also relates to soluble forms of lymphotoxin-β.Soluble lymphotoxin-β peptides are defined by the amino acid sequence oflymphotoxin-β wherein the sequence is cleaved at any point between theend of the transmembrane region (i.e., at about amino acid #44) and thefirst homology region at about amino acid #95. Amino acid sequences oftwo soluble lymphotoxin-β peptides are defined by SEQ ID NO:4 and SEQ IDNO:6. Several additional soluble lymphotoxin-β polypeptides comprise theamino acid sequence as defined by SEQ ID NO:6 with additional amino acidresidues at the 5′ end. The additional residues may comprise the 52amino acid residues as defined by SEQ ID NO:8. The soluble lymphotoxinmay also comprise an amino acid sequence defined by SEQ ID NO:6 plus aportion of SEQ ID NO:8 comprising 1 to 51 of the amino acid residuesbeginning from the 3′ end. Such soluble peptides may include any numberof well known leader sequences at the 5′ end. Such a sequence wouldallow the peptides to be expressed in a eukaryotic system. [See, e.g.,Ernst et al. U.S. Pat. No. 5,082,783 (1992)].

[0044] The polypeptide complexes of this invention comprise a firstpolypeptide comprising an amino acid sequence selected from the group ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and the sequence defined by SEQID NO:6 plus the entirety or a portion of SEQ ID NO:8 as describedabove, complexed with a second polypeptide selected from the same groupand/or with a second polypeptide such as: lymphotoxin-α, native human oranimal lymphotoxin-α, recombinant lymphotoxin-α, soluble lymphotoxin-α,secreted lymphotoxin-α, or lymphotoxin-α-active fragments of any of theabove.

[0045] The novel LT-β peptide forms a complex with LT-α and formscomplexes with other LT-β subunits (e.g., (LT-β)₂ LT-α complexes). Thesecomplexes may be cell associated or not associated with a cell, and maycomplex with other type II membrane proteins sharing common homologyregions as described above.

[0046] The polypeptide complexes are recognized by polyspecific antiserathat recognize recombinant LT-α, suggesting that the complex exhibitsLT-α epitopes. These antisera include a commercial anti-LT-α monoclonalantibody (Boehringer Mannheim), and polyspecific antisera raised againstrecombinant lymphotoxin-α (rLT-α) expressed in transfected ChineseHamster Ovary (CHO) cells. Polyclonal antisera that recognize thesecomplexes also block the mixed lymphocyte reaction (MLR), but amonoclonal anti-rLT-α antibody that recognizes soluble LT-α do not blockthe MLR. The complexes of the present invention thus appear to play animportant role in T cell activation. We also expect these complexes tohave T cell regulatory activities and cytotoxic activities similar tothose of soluble LT-α or TNF.

[0047] This invention also relates to DNA consisting essentially of DNAsequences that code for the polypeptides comprising the amino acidsequences described above, recombinant DNA molecules characterized bythat DNA, hosts selected from the group of unicellular hosts or animaland human cells in culture transfected with that DNA, and recombinantmethods of using that DNA and those recombinant DNA molecules and hoststo produce the polypeptides coded thereby.

[0048] More specifically, this invention relates to an isolated DNAsequence comprising the nucleotide sequence as defined by SEQ ID NO: 1.

[0049] This invention also relates to polypeptides encoded by thatsequence, DNA sequences that hybridize with that DNA sequence that codefor a polypeptide that is substantially homologous with lymphotoxin-β,and degenerate DNA sequences comprising nucleotide sequences that codefor lymphotoxin-β.

[0050] This invention also relates to DNA sequences that code forsoluble lymphotoxin-β peptides. These DNA sequences are defined by SEQID NO:3 and SEQ ID NO:5. This invention also relates to severaladditional soluble lymphotoxin-13 peptides that are coded for by the DNAof SEQ ID NO:5 plus several additional nucleotide triplets at the 5′end. The additional nucleotide triplets may comprise the 52 triplets asdefined by SEQ ID NO:7. The soluble lymphotoxin peptide may also beencoded by SEQ ID NO:5 plus a portion of SEQ ID NO:7 comprising 1 to 51nucleotide triplets beginning from the 3′ end.

[0051] This invention also relates to DNA sequences that hybridize toany of the sequences identified above that code on expression forlymphotoxin-β or soluble lymphotoxin-β peptides. This invention alsorelates to degenerate nucleotide sequences that code for lymphotoxin-βor a soluble lymphotoxin-β peptide, and to DNA sequences that code forpolypeptides that are substantially homologous with a solublelymphotoxin-β peptide.

[0052] Lymphotoxin-β was identified, isolated and characterized usingthe techniques described below:

[0053] Flow Cytofluorometric Analysis

[0054] First we demonstrated the expression of LT-α epitopes on thesurface of T cells using flow cytofluorometric analysis. We observedthat human peripheral blood mononuclear cells activated with OKT3monoclonal antibody demonstrated expression of LT-α epitopes by reactingwith anti-rLT-α antisera. Only anti-rLT-α antisera, not anti-rTNFantisera, bound to OKT3-stimulated primary T cells.

[0055] We also observed that a human T cell hybridoma, II-23.D7 [C. Wareet al., “Human T Cell Hybridomas Producing Cytotoxic Lymphokines:Induction of Lymphotoxin Release And Killer Cell Activity By Anti-CD3Monoclonal Antibody Or Lectins And Phorbol Ester,” Lymph. Res., 5:313(1986)], secreted LT-α upon PMA stimulation and also expressed surfaceLT-α-related epitopes upon PMA stimulation. We also demonstrated thatPMA-activated II-23.D7 cells were able to remove LT-neutralizingantibodies from the rabbit anti-rLT-α antisera, while control U937cells, which lack all surface LT-α forms, were not. We further ruled outthe possibility that the rabbit anti-rLT-α antisera had bound(complexed) with rabbit LT-α (the resulting complexes subsequentlybinding to cellular LT-α/TNF receptors on the II-23.D7 cells) bysaturating the cellular receptors with excess soluble TNF or LT-α andobserving that this had no effect on the staining. These assaysdemonstrate that the LT-α-related epitopes on this hybridoma aregenuinely related to LT-α.

[0056] We also observed that pretreatment of the antisera with excessrLT-α blocked the ability of the antisera to stain II-23.D7 cells, whilepretreatment with rTNF had no effect. This assay demonstrated thespecificity of the antisera for LT-α-related epitopes.

[0057] Trypsinization of the stimulated II-23.D7 cells prior to stainingled to loss of the signal, demonstrating that the epitopes recognized bythe antisera were proteins.

[0058] We also demonstrated that CHO-derived contaminants did notcontribute to the antisera recognition of induced proteins on thesurface of activated II-23.D7 cells by showing that CHO cells stablytransfected with the LT-α gene, which produce only soluble LT-α, werenot stained by the anti-LT-α antisera.

[0059] Immunoprecipitation

[0060] We further characterized these surface LT-α-related proteins byeither surface iodination (¹²⁵I-labelling) or metabolic labelling(³⁵S-Met or ³⁵S-Cys) of PMA-activated II-23.D7 cells, followed bysolubilization of the plasma membrane with detergent andimmunoprecipitation of the labeled LT-α-related proteins.

[0061] Surface iodination coupled with immunoprecipitation revealed twoproteins recognized by the anti-LT-α antisera: a 25-26 kD formsubsequently referred to as LT-α, and a 31-35 kD form subsequentlyreferred to as LT-β or p33. We observed that neither the preimmune serumfrom the same rabbit nor anti-rTNF rabbit serum immunoprecipitated thesebands from the iodinated, PMA-activated II-23.D7 cells. One dimensionalpartial CNBr peptide mapping of the iodinated, immunoprecipitated bandsshowed that the 25-26 kD form (LT-α) cleaves in a pattern identical tothat of iodinated recombinant LT-α, reinforcing the correlation betweensurface LT-α and soluble LT-α. The iodinated 31-35 kD form (LT-β, orp33) was not cleaved by CNBr, indicating that it is distinct from theknown LT-α gene product.

[0062] We further characterized the LT-α and LT-β proteins by metaboliclabelling of PMA-activated II-23.D7 cells with ³⁵S-methionine or³⁵S-cysteine, followed by immunoprecipitation. The distribution ofcysteine and methionine provides a means of distinguishing between TNFand LT-α and between forms of each with and without their signalsequences [M. Kriegler et al., Cell, 53:45 (1988)]. Secreted TNFcontains cysteine, but not methionine, while secreted LT-α contains onlymethionine and no cysteine residues. LT-α, however, has one cysteineresidue in its signal sequence, while TNF contains two methionineresidues in its signal sequence.

[0063] We labeled separate cultures of PMA-treated II-23.D7 cells witheither ³⁵S -methionine or ³⁵S-cysteine and precipitated immunoreactiveproteins from the culture media and the cells. Subsequent SDS-PAGEanalysis of the immunoprecipitates from the culture media of cellslabeled with ³⁵S-methionine revealed a 25 kD form of LT-α while theimmunoprecipitates from the culture media of cells labeled with³⁵S-cysteine did not, a pattern expected for secreted LT-α. Analysis ofthe washed cells showed both the 25-26 kD LT-α form and the 33 kD LT-βform. These results parallelled the membrane-associated forms observedusing surface iodination.

[0064] The 25-26 kD LT-α lacked cysteine, indicating processing of theleader sequence. We also observed that the 33 kD LT-β incorporated both³⁵S-methionine and ³⁵S-cysteine, distinguishing itself as different fromthe 25 kD LT-α form. Typically, LT-α bound to its receptor can becross-linked to the receptor using a chemical linker such as BOSCOES(i.e., (his [2-[succinimidooxy-carbonyloxy]ethyl] sulfone; Pierce,Rockford, Ill.). [J. S. Andrews et al., “Characterization of theReceptor for Tumor-Necrosis Factor (TNF) and Lymphotoxin (LT) on Human TLymphocytes,” J. Immunol., 144:2582 (1990)]. We observed that whensurface iodinated II-23.D7 cells were treated with a cross-linkingagent, there was no association of either the 25-26 kD LT-α or the 33 kDLT-β related form with an additional membrane protein. This assaydemonstrated that receptor binding is not the mechanism by which LT-αand LT-β remain associated with the cell membrane. Sequence analysis ofLT-β showed no relationship to either of the two TNF-receptor forms [C.Smith et al., Science, 248:1019 (1990); T. J. Schall et al., Cell, 61:61(1990)].

[0065] Affinity Chromatography

[0066] Further characterization of the LT-β and LT-α proteins on thesurface of II-23.D7 cells was obtained through affinity chromatography.We observed that LT-α/LT-β complex on the surface of PMA-treatedII-23.D7 cells bound to lentil lectin, indicating a glycoproteinstructure for each form. Hence a lentil lectin chromatography step wasused as a purification step prior to antisera affinity chromatography.We bound detergent-solubilized PMA-treated II-23.D7 proteins to lentillectin sepharose and eluted with α-methyl mannoside. We prepared bothcontrol IgG and anti-LT-α-IgG affinity columns to accurately assessthose proteins specifically recognized by the anti-LT-α antiserum. Wethen applied the proteins that bound to lentil lectin to these columns.We observed that low pH elution of the columns led to the release of theLT-β and LT-α proteins from the anti-LT-α affinity column. SDS-PAGEanalysis of the eluate closely resembled the SDS-PAGE analysis ofimmunoprecipitated proteins from surface iodinated PMA-treated II-23.D7cells. This comparison demonstrated that similar proteins were purifiedby the two methods.

[0067] We observed that during affinity purification, the −25 kD LT-αform appeared to be cleaved to a 19-20 kD form, or a “des 20” form. Theoriginal isolation of natural LT-α from the RPMI 1788 tumor cell line[B. Aggarwal et al., “Primary Structure of Human Lymphotoxin DerivedFrom 1788 Lymphoblastoid Cell Line,” J. Biol. Chem., 260:2334-2344(1985)] also yielded an N-terminally cleaved 20 kD LT-α form. One of themethionines is lost in this “des-20” natural LT-α form, producing adifferent CNBr cleavage pattern from the intact molecule.One-dimensional CNBr digests of the affinity-purified LT-α proteindemonstrated a cleavage pattern that is consistent with the truncatednatural LT-α form, and we concluded that the affinity-purified “des-20”LT-α form probably results from a similar cleavage as observed with thenatural LT-α “des-20” form.

[0068] We further observed that LT-β generates a doublet upon partialCNBr cleavage. The cleavage pattern generated by the LT-β proteindemonstrated that methionine residue(s) were present, and at least onemethionine was within 5-20 residues from either the C- or N-terminus(methionine residues within 1-5 residues of either end would not bedetected when cleaved using this mapping technique). This patternsuggested that LT-β does not contain the entire known LT-α sequence.

[0069] We observed that the LT-β protein is also expressed byantigen-activated primary cytotoxic T lymphocyte clones. Metaboliclabelling of these cells followed by immunoprecipitation with anti-rLT-αrevealed LT-β along with small amounts of LT-α. These resultsdemonstrated that LT-β is made by primary T cells as well as by theII-23.D7 hybridoma.

[0070] Initial Purification of the LT-β and LT-α Proteins

[0071] We purified these LT-α and LT-β proteins using the followinggeneral steps. We first added phorbol myristic acetate (PMA) to II-23.D7cells. After 24 hours we harvested the cells and washed them with coldserum-free RPMI medium. To the chilled cell pellet we added ice-coldlysis buffer (HEPES, NP-40, EDTA, NaCl, and sodium azide) to whichbenzamidine, phenyl methyl sulfonyl chloride (PMSF), and N-ethylmaleimide (NEM), soybean trypsin inhibitor, pepstatin and aprotinin hadbeen freshly added. We homogenized the cells gently in a Douncehomogenizer and centrifuged the lysate. We centrifuged and collected thesupernatant. We loaded the supernatant onto a lentil-lectin sepharosecolumn equilibrated in lysis buffer to which we had added CaCl₂ andMnCl₂. We washed the column with lysis buffer with CaCl₂ and MnCl₂ andthen eluted with lysis buffer containing a-methyl mannoside. We pooledthe eluate fractions and loaded directly onto a rabbit nonspecific IgGsepharose affinity column which was directly connected to a rabbitanti-rLT-α sepharose affinity column. We washed both columns with thesame lysis buffer with EDTA followed by lysis buffer wherein the NP-40had been replaced with MEGA-8 (Octanoyl-N-methyl glucamide,Boehringer-Mannheim). We eluted the washed columns individually with asolution of 5 mM MEGA-8, 50 mM glycine, NaCl, benzarnidine, and EDTA asdescribed in Browning et al., “Lymphotoxin And An Associated 33-kDaGlycoprotein are Expressed On The Surface Of An Activated Human T-CellHybridoma,” J. Immunol. 147:1230-1237 (1991). The first fractionsfollowing the pH shift were pooled, lyophilized and resuspended in waterwith SDS, and dialyzed against a solution of HEPES and SDS. We dried thedialyzed fractions on a speed-vac and resuspended in water. We mixedaliquots with Laemmli loading buffer and electrophoresed on SDS-PAGE. Wevisualized proteins by silver staining.

[0072] We observed that LT-α epitope(s) are present on the surface ofthe II-23.D7 T cell hybridoma only following cell activation such asoccurs with PMA treatment. In contrast, when present on primary T-cells,PMA treatment leads to loss of the surface antigen. Additionally, wefound that rabbit polyclonal antisera to either recombinant LT-α(produced in CHO cells) or natural LT-α (e.g., Genzyme, Boston, Mass.)recognized the LT-α epitope(s).

[0073] We have also observed that our antisera recognizing the LT-α/LT-βcomplex blocks the MLR, whereas a particular monoclonal antibodyrecognizing a soluble LT-α does not [M. Shalaby et al., J. Immunol.,141:499 (1988)]. The LT-α/LT-β complex of the invention, therefore, maybe a mediator in T cell activation.

[0074] The presence of LT-β with LT-α in immunoprecipitates from celllysates suggested that either LT-β is antigenically related to LT-α orthat LT-β is bound to LT-α or both. To address this issue 25 kD and 33kD bands from ³⁵S-methionine labeled cells were immunoprecipitated withrabbit polyclonal anti-rLT-α serum, eluted from excised gel slices andsubjected to reimmunoprecipitation with either anti-rLT-α polyclonalserum or anti-rLT-α mAb. LT-α, but not LT-β, could be immunoprecipitatedwith either of the anti-rLT-α antibodies, suggesting that LT-β is notantigenically related to LT-α. These observations indicated that LT-β isphysically associated with LT-α.

[0075] To further investigate the hypothesis that surface LT-α and LT-βform a complex, we performed isoelectric focusing (IEF) experimentsunder both denaturing and native conditions, the rationale being that ifLT-α and LT-β are physically associated, then they should focus as acomplex under native conditions but as separate entities underdenaturing conditions. The individual isoelectric points (pI's) for LT-αand LT-β were determined by two-dimensional gel analysis (denaturingconditions) (FIG. 9A). LT-α possesses five charged isomers ranging in pIfrom 6.5 to 7.3, whereas LT-β possesses four charged isomers ranging inpI from 5.5 to 6.0. When focusing was performed under native conditions,however, LT-α and LT-β focused together as a broad band ranging in pIfrom 6.3 to 7.2 (FIG. 10A, lanes 6-8). Therefore, the migration of LT-βwas significantly retarded under native conditions.

[0076] Further Purification and Identification of LT-α and LT-β

[0077] We later purified these LT-α and LT-β proteins using thefollowing general steps. We grew II-23.D7 cells in RPMI medium withfetal bovine serum and we harvested the cells from 50 1 of RPMI andresuspended them in medium and we added phorbol myristoyl acetate (PMA).After activation for 6 hours, we harvested the cells by centrifugationand washed them with Dulbecco's phosphate buffered saline. We suspendedthe final cell pellet in cold lysis buffer and passed the pellet oncethrough a nitrogen cavitator. We centrifuged the lysed cells anddiscarded the supernatant. We extracted the pellet overnight in lysisbuffer with detergent and then centrifuged it again.

[0078] We added the supernatant containing the detergent solubilizedmembranes to affinity resin composed of monoclonal anti-LT-α coupled toAffi-gel (10) and rocked the suspension overnight. We collected theresin into a small column and washed it with HEPES with nonidet P40, andthen with the same buffer with 1% w/v MEGA-8, we eluted the boundproteins with MEGA-8 in glycine buffer and neutralized the fractionsimmediately with Tris base. We determined the presence of LT-β and LT-αin the fractions by SDS-PAGE analysis and silver staining. We pooledfractions containing these proteins and added SDS, and we dialyzed thepool against O.1x laemmli sample buffer (using multiple changes toremove the MEGA-8 detergent). We lyophilized the dialyzed solution todryness and resuspended it in {fraction (1/10)}th the original volume ofwater.

[0079] We ran the sample on an SDS-PAGE gel, blotted onto a ProBlotmembrane and stained with Coomassie blue dye. We excised the LT-β andLT-α bands and loaded them into a protein sequencer. We obtained theN-terminal sequence by Edman degradation. We found the sequence of themembrane associated LT-α band to exactly match that described forsecreted LT-a, i.e., Leu Pro Gly Val Gly Leu Thr Pro Ser (amino acid No.1 to 9.) [P. Gray et al., Nature, 312:121-124 (1984)]. The Edmandegradation analysis revealed that the N-terminal portion of theassociated LT-β protein included two possible amino acid sequences: GlyLeu Glu Gly Arg Gly Gin Arg Leu Gln or Gly Leu Glu Gly Arg Leu Gln ArgLeu Gin. Subsequent DNA analysis using more accurate cDNA techniquesconfirmed that the correct sequence was Gly Leu Glu Gly Arg Gly Gly ArgLeu Gin.

[0080] In each case where a surface LT-α form was detected, we were alsoable to detect LT-β (i.e., in PMA-activated II-23.D7, activated CTLclones, and Hut-78 cells constitutively expressing a surface LT-α form).Because LT-α is secreted from transfected CHO cells in the absence of asurface LT-α form, and because the presence of LT-β is associated withsurface-bound LT-α, we concluded that LT-β complexes with LT-α to targetit to the cell surface. Biochemically, LT-β and LT-α co-migrate on anon-denaturing isoelectric focusing gel, but when the complex isdissociated with urea, the two proteins run separately. [See FIGS. 9A,10A.] These observations have led us to conclude that LT-α and LT-βexist as a complex on the cell surface.

[0081] Identifying DNA Sequences That Code For Lymphotoxin-β And SolubleLymphotoxin-β Peptides

[0082] Lymphotoxin-β was purified by immunoaffinity-chromatography asdescribed above. Direct N-terminal sequencing and in situ trypsindigestion followed by reverse phase HPLC resolution of the digestedpeptides was performed. [See, Abersold et al., “Internal Amino AcidSequence Analysis Of Proteins Separated By One Or Two Dimensional GelElectropherisis After In Situ Protease Digestion On Nitrocellulose,”PNAS, 84:6970-6974 (1987)]. The resulting N-terminal and internaltryptic fragment peptides were then sequenced using conventionalmethods. The sequencing of the N-terminus and internal peptides,designated as T105, T87/88, T110 and T67 are shown in FIG. 13.

[0083] Two antisense 17-mer oligonucleotide probes GTYTCNGGCTCYTCYTC[SEQ ID NO:9] and GTYTCNGGTTCYTCYTC [SEQ ID NO:10], designated 1368 and1369, respectively, were synthesized to match a portion the sequence ofpeptide T-87/T-88 and radiolabelled with ³²p Northern analysis showedthat the probe designated 1368 hybridized strongly to a 0.9-1.1 kb mRNAband that was strongly induced in II-23.D7 cells that had beenpretreated with phorbol ester as previously described.

[0084] A cDNA library in the vector pCDM8 was constructed from poly A+mRNA isolated from II-23.D7 cells induced with PMA for 6 hours. Thelibrary was screened with the labelled oligomer designated 1368 andpositive clones were isolated following washing with 3 Mtetramethylammonium chloride at 50° C. Several (>16) clones containing0.8-0.9 kb inserts were subjected to DNA sequence analysis.

[0085] Clone pCDM8/LT-β-12 contained the coding sequence oflymphotoxin-β as shown in SEQ ID NO: 1. The other clones were identicalexcept for various truncations at the 5′ end. The clone 12 cDNA codesfor a functional lymphotoxin-B. Using standard primer extension methods,three additional codons encoding the amino acid residues—MET GLYALA—were identified. A termination sequence AATAAA at position 862-867was found just prior to the 3′ poly A tract indicating that the entire3′ end had been identified. The protein coding sequence encodes for 240amino acids with a calculated unmodified molecular weight ofapproximately 25,390 kDa.

[0086] The 5′ end of the LT-β DNA sequence identified from the clone 12cDNA and primer extension, ATGGGGGCACTGGGGCTG [SEQ ID NO: 11] revealsthree possible start codons (underlined). [See, e.g., M. Kozak, “AnAnalysis Of Vertebrate mRNA Sequences: Intimations Of TranslationalControl,” J. Cell. Biol., 115, 4:887-903 (1991).] Engineered LT-βpolypeptides and DNA sequences may be derived by cleaving all or part ofthis 5′ sequence and substituting a single start codon.

[0087] This amino acid sequence profile is typical of a type II membraneprotein. Following a short (maximum 17) amino acid N-terminal“cytoplasmic” domain there is an extensive stretch of 30 hydrophobicamino acids which presumably acts as a membrane anchoring domain. Noidentical sequences were found within the available databases. There isone cysteine residue in the extracellular domain and two methionineswithin the last C-terminal 17 amino acids. This is consistent with thevery limited cyanogen bromide cleavage pattern exhibited by thisprotein.

[0088] After the sequence of LT-β was determined, subsequent comparisonrevealed that LT-β is a type-II membrane protein with significanthomology to TNF, LT-α and the CD40 protein. These polypeptides sharefour regions of sequence conservation in the extracellular domain. SeeFIG. 14. Such conservation regions are likely to enable thosepolypeptides to form complexes with each other. [See, e.g., M. Eck andS. Sprang, “The Structure Of Tumor Necrosis Factor-α At 2.6 ÅResolution, Implications For Receptor Binding,” J. Biological Chemistry,264, 29:17595-17605 (1989); E. Jones et al., “Structure Of TumorNecrosis Factors,” Nature, 338:225-228 (1989); M. Eck et al., “TheStructure of Human Lymphotoxin (Tumor Necrosis Factor-1) At 1.9-AResolution, J. Biological Chemistry, 267, 4:2119-2112 (1992); J.Tavernier et al., “Conserved Residues Of Tumor Necrosis Factor AndLymphotoxin Constitute the Framework Of The Trimeric Structure,” Fed.Eur. Biochem. Soc. Lett., 257:2 (1989)].

[0089] Expression of Cloned LT-β

[0090] The pCDM8/LT-β clone 12* or a control plasmid, Clone 4 (pCDM8with a non-functional LT-β cDNA insert), were introduced byelectroporation into CHO dhfr⁻ cells and CHO cells stably transfectedwith human LT-α. After three days, cells were removed with Ca/Mg-freeHank's solution with 5 mM EDTA and stained for FACS analysis asdescribed above using either 10 μg/ml control IgG1 or anti-LTαmonoclonal antibody (Boehringer-Mannheim) followed by labelling of boundimmunoglobulin with either a FITC or phycoerythrin labelled goatanti-mouse preparation.

[0091] In other experiments, COS cells were electroporated with eitherclone 4 or clone 12 LT-β cDNA in pCDM8 in the presence or absence of anequal amount of human LT-α cDNA also in the pCDM8 vector and stained forFACS analysis after three days as above. Only CHO cells expressing LT-αdisplayed surface lymphotoxin upon transfection with a functional LT-βDNA, i.e., clone 12.

[0092] Clone 12 lacks an initiating ATG codon, but does possess severalCTG initiating codons and hence this expression experiment shows thatone or several of the 5′ CTG codons must initiate translation. CTGcodons are known to serve as initiating sites for translation in severaleukaryotic proteins [M. Kozak, J. Cell. Biol., 115, 4:887-903 (1991)].Similar results were observed using the dual transfection system in COScells, such that only COS cells receiving both LT-α and LT-β DNAdisplayed substantial surface LT-α in a FACS analysis.

[0093] Potential Uses of LT-β and LT-α and the LT-α/LT-β complex

[0094] As noted above, there is considerable structural similaritybetween LT-β, LT-α, TNF and the CD40 ligand. LT-β, LT-α, TNF and theCD40 ligand are type II membrane proteins and share at least fourregions of sequence conservation in the extracellular domain.

[0095] In light of this structural similarity, it is of interest thatLT-α is found on the surface of activated lymphocytes in a formidentical to the secreted molecule but complexed with an additionalintegral membrane protein presumably anchoring LT-α to the surface. Webelieve that this unique complex, now determined to be LT-α/LT-β,represents a more relevant form of LT-α and imparts specificity relativeto TNF.

[0096] The existence of a heterometric complex of lymphokines, whileunique to the immune system is reminiscent of signalling molecules inother areas, e.g, the PDGF and inhibin/activin heteromeric complexes.Delineation of the LT-α/LT-β complex poses the possibility ofimmunoregulatory activities unique to the complex which cannot bemimicked by the LT-α homotrimer. The complex may bind to a uniquereceptor or receptor chain combinations leading to a high affinityinteraction and biologically relevant signalling. The hypothesis of anLT-α/LT-β interaction with a unique receptor complex could account forthe relatively poor activity of the LT-α homotrimer relative to TNF inmany systems, an observation which cannot be explained by studies on thetwo known TNF receptors. [T. Schall et al., Cell, 61:361-370 (1990); C.Smith et al., Science, 248:019 (1990)].

[0097] The tethering of soluble LT-α to the cell surface viacomplexation with LT-β indicates that cell-cell contact specificsignalling through LT-α/LT-β may be an important aspect of immuneregulation. Because the TNF and related LT-α forms are secreted webelieve that LT-β may also be secreted. This may be verified in studiesusing anti-LT-β monoclonal antibodies. Such antibodies may also be usedto determine whether LT-β homo-oligomers occur naturally.

[0098] In general, LT-α and TNF exhibit qualitatively the same spectraof activities, and LT-α and TNF are believed to interact with the sameset of receptors (designated the 55 and 80 kD TNF receptors). [C. Smithet al., Science, 248:019 (1990); T. Schall et al., Cell, 61:361 (1990)].Nonetheless, the quantitative patterns of biological potency exhibitedby LT-α and TNF are dramatically different, with LT-α often being muchless potent than TNF [see, e.g., Browning et al., J. Immunol., 143:1859(1989)]. These observations are difficult to reconcile with the existingreceptor binding data. It is possible that the LT-α/LT-β complex impartsunique properties on LT-α such that it now interacts with other as yetundefined receptors. In this case, a LT-α/LT-β complex and the othercomplexes of this invention would have unique biological propertiesdistinguishing them from either LT-α or TNF. The LT-α/LT-β complex maybe used to identify and clone such LT-α/LT-β or LT-β specific receptors.Moreover, further use of the complex may reveal novel biologicalactivities.

[0099] Also, while a number of T cell and macrophage cell lines areknown to be infectable by the HIV virus, in practice only a small numberof cell lines have been useful in propagating the virus in tissueculture. For example, the H9 line, a derivative of Hut-78 originallyexploited by Gallo et al. [M. Popovic et al., Science, 224:497-500(1984)], and another human lymphocytic line, C8166, have been valuablefor HIV propagation [M. Somasundaran and H. Robinson, Science,242:1554-1557 (1988)]. It is possible that surface LT-α expression orthe capacity for expression of surface LT-α makes a given cell a goodtarget for HIV proliferation.

[0100] A role for TNF has been proposed in enhancing HIV proliferation[L. Osborn et al., Proc. Natl. Acad. Sci. USA, 86:2336 (1989); Z.Rosenberg and A. Fauci, Immunol. Today, 11:176 (1990); C. Locardi etal., J. Virology, 64:5874 (1990); G. Oli et al., Proc. Natl. Acad. Sci.USA, 87:782 (1990)]. We have found that the II-23.D7 line is infectablewith the HIV strain IIIB, but upon PMA treatment the infection by thevirus is dramatically increased. The Hut-78 cell line was found toconstitutively express a surface LT form, and the C8166 line resemblesII-23.D7 in that surface LT appears following PMA treatment. [Ware etal., J. Immunol., article in press (1992)].

[0101] Considering these results on the infectability of II-23.D7 by HIVand the relationship between infectable cell lines and surface LTexpression, we propose that those lines may be good hosts for HIVinfection and replication because the LT-α/LT-β complex and the otherpolypeptides and complexes of this invention serve a regulatory role. Ithas been demonstrated that the LT-α gene is induced by expression of theHIV transcriptional activator TAT [K. Sastry et al., J. Biol. Chem.,265:20091 (1990)] and, moreover, HTLV-1 infection has also been shown toinduce LT-α expression [N. Paul, et al., J. Virol., 64:5412 (1990); E.Tschachler et al., in Human Retrovirology (Raven Press 1990), W.Blattner, eds., p. 105]. Thus, induction of LT-α by HIV infection andconsequent LT-α/LT-β complex or other complex expression, or inductionof LT-α/LT-β or other complex expression by PMA treatment in cell linescompetent to make these proteins, may serve to enhance viralreplication. For this reason, antibodies or specific binding proteins(e.g., soluble receptors) to the LT-α/LT-β complex or the otherpolypeptide complexes of this invention or to soluble forms of thosecomplexes or to LT-β and the other polypeptides of this invention mayinhibit HIV proliferation or block HIV-induced T cell death.

[0102] Parallels may be drawn between LT-α/LT-β and the CD40 receptorligand pair where signalling from a T cell surface CD40 ligand provides“help” to the B cell via the CD40 receptor. One could thereforepostulate that surface LT-α/LT-β may be a component of T cell regulationof T cells or other cells of hematopoietic lineage such as LAK or NKcells and B-cells. Moreover, this interaction may be dysfunctional insome autoimmune diseases. [See, e.g., R. Watanabe-Fukunaga, Nature,356:314-317 (1992).]

[0103] Furthermore, a cell surface protein designated as the Fas antigenhas been shown to have considerable structural homology with a number ofcell-surface receptors including TNF, NGF and the CD40 protein. The Fasantigen has been implicated in mediating apoptosis, a process alsoreferred to as programmed cell death [R. Watanabe-Fukunaga, Nature,356:314-317 (1992); N. Itoh et al., Cell, 66:233-243 (1991)]. A strainof mice that demonstrates defects in the Fas antigen develop a systemiclupus erythematosus-like autoimmune disease. This suggests that thestructurally similar LT-β or LT-α/LT-β complexes may also play a role inmediating systemic lupus erthyematosus and, therefore, intervention inthis pathway may be clinically useful in treating various autoimmunediseases. Alternatively, LT-β or an LT-α/LT-β complex may be involved ininducing programmed cell death through a cell-cell contact dependentmechanism. The emergence of this family of TNF related ligands tocomplement the already extensive family of TNF/NGF type receptorssuggests the existence of an additional array of important regulatoryelements within the immune system.

[0104] LT-β or a LT-β/LT-α complex may similarly play a role insuppressing the immune system and may be potentially useful in treatingallergy and inducing tolerance.

[0105] The location of the TNF/LT locus in the MHC region of the genomehas led workers to examine linkage to various autoimmune disease,especially insulin-dependent diabetes melitis. [See, e.g., F. Pociot etal., “A Tumor Necrosis Factor Beta Gene Polymorphism In Relation ToMonokine Secretion And Insulin-Dependent Diabetes Mellitus,” Scand. J.Immunol., 33:37-49 (1991); K. Badenhoop et al., “TNF-α GenePolymorphisms In type 1 (Insulin-Dependent) Diabetes Mellitus,”Diabetologia, 32:445-448 (1989).] Because we found that the LT-β gene islocated next to the TNF/LT locus, it is possible that the LT-β gene orits receptor may be involved in this autoimmune condition. Hence, LT-βor its receptor or antibodies to LT-β may comprise a replacement therapyin this form of diabetes.

[0106] As discussed above, the LT-β polypeptide, and the polypeptidecomplexes of this invention, are expected to have a number of potentialuses including anti-tumor, T cell activating, or T cell suppressingapplications, application involving the treatment of systemic lupuserythematosus, as well as uses in anti-inflammatory compositions andmethods. DNA sequences coding for LT-β polypeptides, recombinant DNAmolecules including such DNA sequences, and unicellular hosts and animalor human cells in culture transfected with such recombinant DNAmolecules may then be employed to produce large amounts of thepolypeptides of this invention, substantially free from other humanproteins, for use in the compositions and therapies noted above.

[0107] Lymphocytes expressing on their surfaces the polypeptidecomplexes of this invention, and preferably an LT-α/LT-β complex,represent a subset of lymphocytes that may have enhanced abilities tokill tumor cells. As such, this subset would be useful in LAK(lymphokine-activated killer) cell or TIL (Tumor InfiltratingLymphocyte) cell therapies. [H. Thomas, K. Sikora, “BiologicalApproaches to Cancer Therapy,” Jour. Int. Med. Res., 17:191 (1989)]. TILimmunotherapy may be improved by gene transfer techniques. Recombinantgenes for LT-β and related polypeptides based thereon will be usefultherapeutically, for example in TIL therapy, where a LT-β gene, eitherwith or without an LT-α gene, is introduced into T cells isolated from atumor and introduced to the patient. More preferably, the cells aretaken from the patient, transfected with a DNA sequence encoding onexpression a polypeptide of this invention, before or after thattransfection incubated with a lymphokine, preferably IL-2, and returnedto the patient. The transfected T cells (now expressing LT-β and alsoconsequently complexing LT-α) home in on the tumors from which they wereremoved, where the tumorcidal action of LT-α is delivered directly tothe tumors. Likewise, it is contemplated that a LT-β gene introducedinto LAK cells would increase the number of surface complexes on thecells and enhance their activity. Alternatively, introduction of theLT-β gene into a patient's tumor cells may be useful in creating a tumorvaccine in which the LT-β modified tumor would trigger an enhancedimmune response to the tumor itself. [See, e.g., W. F. Anderson,Science, 256:808-813 (1992)].

[0108] Antibodies or antibody derivatives to the polypeptides andpolypeptide complexes of this invention are also useful in conventionalimmunological methods, e.g., panning or flow cytofluorometric sorting,to enrich for this cell population. [L. J. Wysocki and V. L. Sato,“Panning for Lymphocytes: A method for Cell Selection,” PNAS 75:2844(1978)].

[0109] It is also contemplated that the polypeptides and polypeptidecomplexes of this invention, or fragments or derivatives thereof, willbe useful in cell regulatory or therapeutic applications similar tothose in which lymphotoxin-α and tumor necrosis factors are used.

[0110] The compositions of this invention will be administered at aneffective dose to treat the particular clinical condition addressed.Determination of the particular dose for a given application is wellwithin the skill of the art taking into consideration, for example, thecondition and weight of the patient, the extent of desired treatment andthe tolerance of the patient for the treatment. Administration of thecomplexes and polypeptides of this invention, or perhaps peptidesderived or synthesized therefrom or using their amino acid sequences,including isolated and purified forms of the polypeptides, or theirsalts or pharmaceutically acceptable derivatives thereof, may be via anyof the conventionally accepted modes of administration of agents whichexhibit anti-tumor, T cell-activating, T cell-suppressing oranti-inflammatory activity.

[0111] The compositions used in these therapies may also be in a varietyof forms. These include, for example, solid, semi-solid and liquiddosage forms, such as tablets, pills, powders, liquid solutions orsuspensions, suppositories, injectable and infusible solutions, andgenetic therapy. The preferred form depends on the intended mode ofadministration and therapeutic application. Modes of administration mayinclude oral, parenteral, subcutaneous, intravenous, intralesional ortopical administration. The compositions also will preferably includeconventional pharmaceutically acceptable carriers and may include othermedicinal agents, carriers, genetic carriers, adjuvants, excipients,etc., e.g., human serum albumin or plasma preparations. Preferably, thecompositions are in the form of a unit dose and will usually beadministered one or more times a day.

[0112] The following are examples which illustrate the LT-β and theLT-α/LT-β complex of this invention and the methods used to characterizethem. These examples should not be construed as limiting: the examplesare included for purposes of illustration and the present invention islimited only by the claims.

EXAMPLES

[0113] We used the following experimental procedures in the examples:

[0114] Antisera

[0115] Recombinant human LT-α(rLT-α) was expressed and secreted by astably transfected chinese hamster ovary (CHO) cell line into serum-freeconditioned media. We purified the secreted rLT-α from the serum-freeconditioned media by a series of Sepharose S, lentil lectin-sepharoseand FPLC Mono Q column chromatography steps. The properties of the CHOcell-derived r LT-α preparation have been described. [J. Browning etal., J. Immunol., 143:1859 (1989)]. We immunized two rabbits (4 and 5)by a lymph node procedure [M. Sigel et al., “Production Of Antibodies ByInoculation Into Lymph Nodes,” Met. Enz., 93:3 (1983)] with 25 μg ofnative r LT-α in complete Freund's adjuvant. A third rabbit (6) wasimmunized via the same route with 25 μg of denatured r LT-α in completeFreund's adjuvant. We prepared denatured r LT-α by SDS-PAGE followed byelectroelution into 0.1% SDS-carbonate buffer.

[0116] Using the above methods, three anti-rLT-α antisera weregenerated, two directed against native rLT-α and a third againstSDS-denatured rLT-α. The antisera raised by immunization with nativeprotein (rabbits 4 and 5) could neutralize a 50 unit/ml solution at adilution of 1:2000-5000. The serum raised against denatured rLT-α(rabbit 6) lacked neutralizing titer, but was weakly reactive with rLT-αon a Western blot. None of the antisera could neutralize r-human TNF norcould they recognize r-human TNF bound to an ELISA plate except for avery weak titer in the antiserum from rabbit 6. Only antiserum fromrabbit 6 was capable of recognizing rLT in a western analysis.

[0117] We immunized a fourth rabbit with recombinant human TNF. Weprepared the polyclonal anti-rTNF rabbit serum via a classicalimmunization scheme using recombinant human TNF (E. coli derived [D.Weir et al., Handbook Of Experimental Immunology In Four Volumes,Chapter 8 “Immunization Of Experimental Animals”]) in complete Freund'sadjuvant followed by a boost in incomplete Freund's adjuvant. The serumraised against rTNF by immunization had a good neutralizing titer. Aneutralizing monoclonal antibody to TNF has been described [Liang etal., “Production And Characterization Of Monoclonal Antibodies AgainstRecombinant Human Tumor Necrosis Factor/Cachectin,” Biochem. Biophys.Res. Comm., 137:847 (1986)]. Pre-immunization serum was collected fromall animals for use as controls.

[0118] Cell Growth and T cell Activation

[0119] All cells were obtained from the American Type Culture Collection(ATCC), except for the LT-α transfected Chinese hamster ovary (CHO) linethat was described previously [Browning, J. Immunol., 143, pp. 1859-1867(1989)].

[0120] Cells were grown in RPMI 1640 supplemented with 1% glutamine, 10mM HEPES buffer, pH 7.5, penicillin/streptomycin and 10% fetal bovineserum (Hyclone-defined) (designated “complete RPMI”), except for thetransfected CHO cells which were grown in Dulbecco's modified Eagle'smedium supplemented as above. The human T cell hybridoma, II-23, was aresult of a fusion of the human CEM tumor line with activated peripheralT lymphocytes and was further subcloned (II-23.D7) [C. Ware et al.,“Human T Cell Hybridomas Producing Cytotoxic Lymphokines: Induction ofLymphotoxin Release And Killer Cell Activity By Anti-CD3 MonoclonalAntibody Or Lectins And Phorbol Ester,” Lymph. Res., 5, 313 (1986)].Human peripheral blood lymphocytes (PBL) were drawn into heparinizedglass tubes, isolated by Ficoll-Hypaque centrifugation, washed andresuspended in complete RPMI medium. We treated PBL at 2×10⁶ cells/mlwith a 1:500 dilution of OKT3-conditioned medium (⁻2 ng/ml final) in thepresence of 1 μg/ml indomethacin and, in some experiments, with 10 ng/mlrIL-2 (Biogen, Inc., Cambridge, Mass.)). Human CTL-clones were generatedas described [L. Green et al., “Cytotoxic Lymphokines Produced By ClonedHuman Cytotoxic T Lymphocytes,” J. Immunol., 135:4034 (1985)] andactivated either with irradiated stimulator cells (antigen) or acombination of anti-CD2 monoclonal antibodies (T11₂+T11₃) provided by E.Reinherz.

[0121] Flow Cytometry

[0122] We resuspended cells in RPMI 1640 medium with 10% fetal bovineserum (FBS), 0.1% sodium azide and 0.1 mg/ml human IgG at 0° C.Following preincubation with the human IgG, we added additional mediacontaining the desired antisera. Typically the cells were incubated witha final dilution of the anti-rLT-α and anti-rTNF sera of 1:200 for 60-90min. We washed cells twice with Dulbecco's phosphate buffered saline(PBS) and then incubated them with a 1:500 dilution offluorescein-labeled goat anti-rabbit IgG (Cappel Durham, N.C.) in theabove medium for a minimum of 60 min. Cells were then washed once andeither analyzed directly or, in some cases, analyzed following fixationfor 10 min. at 0° C. with 0.5% paraformaldehyde. We performed two coloranalyses as above, except that we added phycoerythrin labeled leu-4,leu-2, leu-M3 or leu-16 or leu-19 (Becton-Dickinson, Mountain View,Calif.) at the second antibody stage. The comparison of surface-boundLT-α with IL-2 receptor levels was done with separate single coloranalyses with fluorescein-labeled anti-IL-2 receptor (CD25) antibody(Becton-Dickinson, Mountain View, Calif.). Analyses were performed witha FACStar instrument (Becton-Dickinson).

[0123] Adsorption of Neutralizing Anti -rLT-α Antibodies by ActivatedII-23.D7 Cells

[0124] We stimulated II-23.D7 and U937 premonocytic cells at 1×10cells/ml for 8 hours with 10 ng/ml of PMA in complete RPMI medium. Wewashed the cells (1×10⁸) three times in medium and aspirated thesupernatant to obtain a dry pellet. The cells were then resuspended in 1ml of medium containing a 1:1000 dilution of anti-rLT-α serum (fromrabbit 4) and incubated on ice for 1.5 hours with mixing. Cells werecleared from the antiserum by centrifugation. We mixed the absorbedantiserum (both pre- and post-immune) with an equal volume (50 μl) ofmedium containing 15 U/ml of rLT-α and incubated for 20 minutes at roomtemperature. The mixtures were diluted serially into medium and added toL929 cells (in 0.1 ml) and incubated a further 24 hours. We assessedcell viability by the MTT assay as described [L. Green et al., “RapidColorimetric Assay For Cell Viability: Application To The Quantitationof Cytotoxic And Growth Inhibitory Lymphokines” Jour. Immunol. Meth.,70:257-268 (1984)].

[0125]³⁵S-Methionine or ³⁵S-Cysteine Metabolic Labelling of T cells

[0126] We transferred cells into either cysteine-free or methionine-freeRPMI 1640 supplemented with penicillin/streptomycin, glutamine, 10 mMHEPES pH 7.5, 10% v/v dialyzed FBS and 2% v/v conventional RPMI (coldcarrier addition). We adjusted the cell concentration to 2-3×10⁵cells/ml and added ³⁵S-methionine or ³⁵S-cysteine to the appropriatemedium to a level of 100-200 μCi/ml. In the case of freshly activatedPBL, the supernatants were gently removed, and the cells werecentrifuged, resuspended in labelling medium and added back to theoriginal adherent population. Following a 12-18 hour labelling period,we washed and lysed the cells as described below. With the PBL, cellswere removed by pipetting and the adherent population partially removedby treatment with 5 mM EDTA in PBS.

[0127] Immunoprecipitations

[0128] To 0.2-0.5 ml of labeled cell lysate we added 2-4 μl of rabbitserum. The sample was left for 1-2 hours at 4° C. We then added a 60 μlaliquot of a 60-75% suspension of washed Protein A sepharose (Pharmacia,Piscataway, N.J.) and rocked the sample for 6-18 hours at 4° C. Wewashed the Protein A sepharose pellets 3 times with 1% NP-40 incalcium/magnesium free PBS and resuspended them in 50 μl of Laemmli SDSloading buffer. Typically a single lysate sample was cycled throughsequential immunoprecipitations with preimmune anti-rLT-α serum,anti-rTNF antiserum and finally post immune anti-rLT-α antiserum. In oneset of experiments, we added 5 mM CaCl₂ and MnCl₂ to the lysate and thelysate was rocked overnight with 75 μl of 75% suspension of washedlentil lectin-sepharose. The sepharose was washed twice with NP-40/PBSand then eluted with 3 consecutive additions of 75 μl of 1% NP-40/PBSwith 0.25 M a-methyl mannoside. We subjected the pooled washes to theimmunoprecipitation protocol.

[0129] Rabbit Anti-rLT Affinity Column

[0130] We purified the immunoglobulin fraction from the anti-rLT serum(from rabbit 4) using Protein A sepharose with acid pH elution. Theeluted IgG-containing fractions were dialyzed against PBS andconcentrated by amicon filtration. The anti-rLT-α-IgG solution (15 ml of6 mg/ml) was coupled to 8 ml of Affi-gel 10 resin (Biorad, Richmond,Calif.) as per instructions. We prepared an identical affinity columnusing nonspecific rabbit IgG (Cappel, Durham, N.C.). Both columns werewashed with PBS, 1 M acetate pH 3.0 with 1% NP-40 and finally with lysisbuffer lacking protease inhibitors.

[0131] Initial Purification of LT-β and LT-α

[0132] We grew II-23.D7 cells (15 1) to a density of 5×10⁵ cells/ml andadded phorbol myristic acetate (PMA) to give a final concentration of 25ng/ml. After 24 hours, we harvested the cells and washed them into coldserum-free RPMI medium. To the chilled cell pellet containing 7×10⁹cells we added 100 ml of ice-cold lysis buffer (50 mM HEPES pH 7.5, 1%v/v NP-40, 2 mM EDTA, 0.15 M NaCl and 0.1% sodium azide) to which 5 mMbenzamidine, 1 mM phenyl methyl sulfonyl chloride (PMSF) and 0.25 mMN-ethyl maleimide (NEM), 10 ìg/ml soybean trypsin inhibitor, 0.7 μg/mlpepstatin and 10 μg/ml aprotinin had been freshly added. We gentlyhomogenized the cells in a dounce homogenizer and centrifuged the lysateat 10,000×g for 10 minutes. We centrifuged the supernatant at 60,000×gfor 90 minutes and collected the supernatant.

[0133] To the supernatant from the high speed centrifugation we added 5mM CaCl₂ and 5 mM MnCl₂. The supernatant was then loaded onto a 20 mllentil-lectin sepharose column (Pharmacia, Piscataway, N.J.)equilibrated in lysis buffer plus CaCl₂ and MnCl₂. We washed the columnwith lysis buffer (with CaCl₂ and MnCl₂) and then eluted the column withlysis buffer containing 0.25 M a-methyl mannoside.

[0134] We pooled the lentil lectin eluate fractions to give a volume of50 ml and loaded them directly onto a 2 ml rabbit nonspecific IgGsepharose affinity column. We connected this column directly to a 2 mlrabbit anti-rLT-α sepharose affinity column. We washed both columns withthe same lysis buffer with EDTA, followed by lysis buffer wherein the 1%NP-40 had been replaced with 1% w/v MEGA-8 (Octanoyl-N-methyl glucamide,Boehringer-Mannheim, Indianapolis, Ind.).

[0135] We eluted the washed columns individually with 1% MEGA-8, 50 mMglycine pH 2.5, 0.05 M NaCl, 5 mM benzamidine, and 2 mM EDTA. We pooledthe first 20 ml following the pH shift, lyophilized the pool andresuspended it in 1 ml of water with 0.05% SDS, and dialyzed it against10 mM HEPES pH 7.5, 0.05% SDS and 0.1% MEGA-8. We dried the dialyzedfractions on a speed-vac and resuspended them in 0.15 ml of water. Wemixed aliquots with Laemmli loading buffer and electrophoresed them onSDS-PAGE. The LT-β and LT-α proteins were visualized by silver staining.

[0136] Iodination of II-23.D7 Cell Surface

[0137] Either control or PMA-induced II-23.D7 cells were washedextensively in calcium/magnesium-free PBS, treated with 1 mM PMSF and0.25 mM NEM and then washed twice. To a 12×75 mm glass tube that wascoated with 50 μg of iodogen (Pierce) we added 0.3 ml of cells (1×10⁷total) and 1-2 mCi of ¹²⁵sodium iodide. Cells were left with periodicswirling for 25 minutes at room temperature, washed 3 times in PBS with10% FBS and resuspended in lysis buffer as described above. We thenremoved the nuclei with a 2 minute centrifugation in an Eppendorfcentrifuge. We then centrifuged the supernatant an additional 15minutes. The cleared supernatant was subjected to theimmunoprecipitation protocol.

[0138] 1-Dimensional CNBr Peptide Mapping

[0139] We electrophoresed samples on a 12% acrylamide SDS-PAGE Laemmlisystem gel for a short distance and excised the appropriate gelsections. We soaked the gel slices for 1 hour in 1.0 ml of 0.1 N HCl,0.2% 2-mercaptoethanol with 15 μl of 700 mg/ml fresh CNBr in 90% formicacid. The slices were then removed and washed for 5 minutes with 0.1 MTris-Cl pH 8.0, 5 min with 25 mM Tris-Cl pH 8.0 and finally 10 min withlx Laemmli SDS-PAGE loading buffer. We loaded the slices onto a 15%SDS-PAGE Laemmli gel with a 12% acrylamide stacking gel. We visualizedthe peptide bands by silver staining or autoradiography of the driedgel.

[0140] Reimmunoprecipitation

[0141] Reimmunoprecipitation of SDS-PAGE-separated antigens was carriedout by excising labeled bands from gels, rehydrating them for 10 minutesin TBS, 0.2% SDS, and then dicing the gel slices into small pieces. Theproteins were eluted by incubation in 1 ml TBS, 0.2% SDS for 8 hours atroom temperature, with rotation. After elution the gel pieces wereremoved by centrifugation, and NP-40 was added to the supernatant to afinal concentration of 2%. The eluted proteins were thenimmunoprecipitated as above, and reanalyzed by SDS-PAGE.

[0142] Isoelectric Focusing (IEF)

[0143] Two-dimensional IEF was performed essentially as described by P.H. O'Farrell [J. Biol. Chem., 250:4007-4021 (1975)]. ¹²⁵I-labeledantigens were immunoprecipitated from II-23.D7 cell extracts, and theimmunoprecipitated proteins were eluted by heating at 100° C. for 5minutes in 100 μl O'Farrell sample buffer containing 9.5 M urea. Theeluted proteins were then focused (first dimension) on 14 cm×3 mm tubegels possessing a 2% final concentration of ampholines (range pH 3-10,Sigma) at room temperature for 16 hours at a constant voltage (400 V).The second dimension was 12% SDS-PAGE.

[0144] For IEF under native (non-denaturing) conditions, 125I-labeledcell extracts were focused directly on tube gels identical to thoseabove except for the presence of urea. The labeled extract (200 μlvolume) was centrifuged at 100,000×g (30 psi, airfuge) for 10 minutesprior to loading onto the tube gel. The focusing was performed at 4° C.under the same conditions as described above. The tube gel was thenremoved and sliced into 1 cm sections, and the proteins were eluted byincubating each slice in 1 ml TBS, 2% NP-40, 2 mM PMSF for 8 hours atroom temperature, with rotation. The supernatants containing the elutedproteins were then immunoprecipitated and analyzed by SDS-PAGE. The pHgradients for both the denatured and native tube gels were determined bymeasuring the pH's of individual slices from gels run in parallel.

[0145] T Cell Proliferation Assays

[0146] We isolated and resuspended PBL in complete RPMI as describedabove except for the substitution of fetal bovine serum with 10% humanautologous serum, 1 μg/ml indomethacin and 50 U/ml polymyxin B. In theMLR experiments, autologous serum was the responder's serum. Weirradiated stimulator cells from a different donor with 3000 rads. Wepreheated rabbit sera for 1 hour at 56° C., and diluted and sterilefiltered the sera prior to use in proliferation assays. Cells (1×10⁵total) in 0.2 ml in a round bottom 96-well plate were treated witheither 5 μg/ml phytohemagglutinin, 1-2 ng/ml OKT3 or 1.5-2×10⁵irradiated stimulator cells in the presence or absence of variousantisera or cytokines. After 3 days (PHA or OKT3 activation) or 5 days(MLR), cells were pulsed with ³H-thymidine, harvested and counted.

[0147] Further Purification of LT-α and LT-β

[0148] We grew II-23.D7 cells in RPMI medium with 10% fetal bovine serumand we harvested the cells from 50 1 RPMI medium and resuspended them inmedium at a concentration of 4×10⁶ cells/ml and we added 50 ng/mlphorbol myristoyl acetate (PMA). After activation for 6 hours weharvested the cells by centrifugation and washed them with Dulbecco'sphosphate buffered saline. We suspended the final cell pellet of 4×10¹⁰cells in 200 ml of cold lysis buffer (50 mM HEPES buffer, pH 7.0; 0.1 MNaCl, 10 mM EDTA, 5 mM benzamidine, 10 μg/ml each of soybean trypsininhibitor, aprotinin, chymostatin, leupeptin, antipain, 1 μg/mlpepstatin and 1 mM phenylmethyl sulfonyl fluoride) and passed the pelletonce through a nitrogen cavitator. We centrifuged the lysed the cells at40,000 rpm for 60 minutes in a 50.2 Ti rotor and discarded thesupernatant. We extracted the pellet overnight in 120 ml of lysis bufferwith 1% w/v Nonidet P40 detergent and then centrifuged it as above.

[0149] We added the supernatant containing the detergent solubilizedmembrane proteins to 2 ml of affinity resin composed of monoclonalanti-lymphotoxin (anti-tumor necrosis factor-β from Boehringer Mannheim)coupled to Affi-gel 10 (BioRad) and rocked the suspension overnight. Wecollected the resin into a small column and washed it with 50 mM HEPES,pH 7.0 with 1% Nonidet P40, and then with the same buffer with 1% w/vMEGA-8 (Boehringer Mannheim). We eluted the bound proteins with 1%MEGA-8 in 50 mM glycine buffer pH 2.5 and the fractions immediatelyneutralized with Tris base. We determined the presence of p33 and LT inthe fractions by SDS-PAGE analysis and silver staining. We pooledfactions containing these proteins and added SDS to a finalconcentration of 0.1% w/v and we dialyzed the pool against 0.1x Laemmlisample buffer (multiple changes to remove the MEGA-8 detergent). Welyophilized the dialyzed solution to dryness and resuspended it in1/10th the original volume of water. We ran the sample on an SDS-PAGEgel, blotted onto a ProBlot membrane (Applied Biosystems) and stainedwith coomassie blue dye.

[0150] This scheme allows one to purify LT-β to a band on a blot. Itshould be possible for anyone skilled in the art to separate theproteins eluted from the affinity resin by ion exchange chromatography.For example, the complex can be dissociated with urea and the LT-α andLT-β proteins can be separated by, e.g., MONO Q FPLC (Pharmacia) anionexchange chromatography in Tris-Cl buffer pH 8.0 with 1% nonionicdetergent (e.g., MEGA-8, Boehringer-Mannheim) and urea, using a saltgradient elution. This chromatographic technique separates on the basisof differing charges on the proteins. The two proteins are separable inan isoelectric focusing experiment (see, supra) on the basis of chargedifferences, wherein urea is used to dissociate the LT-α/LT-β complex.Such a combination of affinity chromatography, dissociation inurea/nonionic detergent and ion exchange chromatography allowspurification of soluble LT-β or the LT-α/LT-β complex.

[0151] Peptide Sequencing Assays

[0152] We excised the LT-β and LT-α bands from the Problot and loadedthem into a protein sequencer. We obtained N-terminal sequenceinformation by Edman degradation with a model 470A Applied Biosystemssequencer coupled to a 120A PTH amino acid analyzer. LT-β was purifiedby immunoaffinity-chromatography as described above and tryptic fragmentsequence was obtained. [See Abersold et al., “Internal Amino AcidSequence Analysis Of Proteins Separated By One Or Two-Dimensional GelElectrophoresis After In Situ, Protease Digestion On Nitrocellulose,”PNAS:84:6970-6974 (1987)]. That is, protein on the blot was digestedwith trypsin in situ followed by reverse phase HPLC resolution of thedigested peptides. The resulting N-terminal and internal trypticfragment peptides were then sequenced by Edman degradation. Thesequencing of the N-terminal and internal peptides designated as T105,T87/88, T100 and T67 are shown in FIG. 13.

[0153] Construction of Oligonucleotide Probes

[0154] From the sequence of T87/88 the following antisense probes weredesigned: 1368 GTYTCNGGCTCYTCYTC [SEQ ID NO:9] 1369 GTYTCNGGTTCYTCYTC[SEQ ID NO:10]

[0155] and synthesized by standard methods. [See, e.g., J. Sambrook etal., Molecular Cloning, A Laboratory Manual, 2ed. (1989)].

[0156] Preparation of an Induced 11-23 cDNA Library

[0157] We prepared a cDNA sublibrary as follows:

[0158] We stimulated II-23.D7 cells for six hours with 50 ng/ml PMA toensure the presence of LT-β mRNA. We isolated the mRNA from these cellsand reverse-transcribed it into cDNA using techniques well known to theart. [B. Seed and A. Aruffo, “Molecular Cloning Of The CD2 Antigen, TheT-Cell Erythrocyte Receptor, By A Rapid Immunoselection Procedure,”PNAS,84:3365-3369 (1987)]. Using standard procedures, we ligated doublestranded cDNA to a NotI-BstXI linker/adaptor having the followingsequence: 5′ GCG GCC GCT TTA GAG CAC A 3′ [SEQ ID NO:12] 3′ CGC CGG CGAAAT CTC 5′

[0159] We then size-selected the cDNA on a 4.2 ml 5-20% potassiumacetate gradient, 2 mM EDTA, 1 μg/ml ethidium bromide, in a Beckman®SW60 Rotor for 3 hours at 50,000 rpm at 22° C. according to standardmethods. We pooled the cDNA fragments of greater than 500 base pairs.Then we prepared the vector, pCDM8 (a gift from Brian Seed(Massachusetts General Hospital). We digested this plasmid with BstXI.To remove the 400 base pair stuffer fragment we centrifuged the mixtureon a potassium acetate gradient, as above, and isolated the largefragment. We further purified this fragment by agarose gelelectrophoresis, and then ligated the cDNA to the vector. In this way,we created recombinant DNA molecules containing DNA sequences for mRNAexpressed in induced II-23.D7 cells. We used these plasmids to transformE. coli MC 1061 P3. The result was a collection of over 1×10⁶recombinant clones comprising a cDNA library for PMA induced II-23.D7mRNA.

[0160] Screening and DNA Sequencing of Clones

[0161] The pCDM8 II-23.D7 library was screened with ³²P labelledoligomer 1368 and positive clones were isolated following washing with3M tetramethylammonium chloride at 50° C. [J. Sambrook et al., MolecularCloning, A Laboratory Manual, (1989); Jacobs et al., “The ThermalStability Of Oligonucleotide Duplexes In Sequence Independent InTetraalkylammonium Salt Solutions: Application To IdentifyingRecombinant DNA Clones”, Nucleic Acids Research, 16:10:4637-4649(1988)]. Several clones containing 0.9 kb inserts were subject todideoxynucleotide DNA sequence analysis [Id.].

[0162] Expression of LT-β cDNA

[0163] The pCDM8/LT-β clone 12 or a control plasmid, clone 4 (pCDM8 withan irrelevant cDNA insert), was introduced by electroporation into CHOdhfr⁻ and a CHO cell stably transfected with human LT-α. After threedays, cells were removed with Ca/Mg-free Hank's solution with 5 mM EDTAand stained for FACS analysis as described above using either 10 μg/mlcontrol IgG, or anti-LT monoclonal antibody (Boehringer-Mannheim)followed by labelling of bound immunoglobulin with either a FITC orphycoerythrin labelled goat anti-mouse preparation. In otherexperiments, COS cells were electroporated with either clone 4 or clone12 LT-β cDNA in pCDM8 in the presence or absence of an equal amount ofhuman LT-α cDNA also in the pCDM8 vector and stained for FACS analysisafter three days as above.

[0164] Northern Analysis of LT-β Expression

[0165] Poly A+ RNA was isolated from either II-23.D7 cells or peripheralblood mononuclear cells (PMBC) using the Fast-Track™ system provided byInvitrogen. Northern blots were prepared using 2 μg/lane of RNA andelectrophoresis on a formamide gel essentially as described in J.Sambrook et al., Molecular Cloning, A Laboratory Manual, (1989),followed by transfer onto Gene Screen nylon membrane and UVcrosslinking. Blots were probed with random primed BstEII/Xmn-I fragmentof the LT-β cDNA which had been gel purified, or a fragment of humanLT-α or actin. II-23.D7 cells were induced with 50 ng/ml PMA for varyingtimes and both LT-α and LT-β expression was found to be induced. PBMCwere either cultured in RPMI medium alone or in the presence of 1000units/ml of IL-2 or with OKT3 to activate the T-cells.

[0166] Determination of the 5′ End of LT-β

[0167] The 5′ mRNA sequence was determined by primer extension analysis.[B. Wallner et al., Nature 320:77-81 (1986).] Primer extension using anoligonucleotide primer (probe 360-121 5′GACAGTGATAGGCACCGCCAGCAACAA -3′)[SEQ ID NO:13] yielded a roughly 128-130 bp product that upon sequencingusing Maxam and Gilbert methodology [A. Maxam and W. Gilbert,“Sequencing End-Labeled DNA With Base-Specific Chemical Cleavages,”Methods In Enzymology, 65:499 (1988)] showed the transcriptional startsite to be 7-9 bp upstream of the methionine ATG. The expressionexhibited by clone 12 in transient experiments indicates that one orboth of the Leu-4 or Leu-6 start site is functional. To verify the 5′mRNA sequence, a cosmid clone, 031A [Spies et al., Science 243:214(1989)], was digested with several restriction enzymes, electrophoresed,blotted and probed with a BST E2/Xmn-1 fragment of the LT-β cDNA. Thecosmid contained the LT-β gene within a 6 kb EcoR1 fragment which wassubcloned into a pUC derivative called pNN 109 which contained akanamycin resistance gene. Dideoxynucleic acid sequencing gave theentire genomic sequence.

Example 1

[0168] T Cells Express LT-Related Epitopes on Their Surfaces

[0169] Under the conditions described above, we activated humanperipheral mononuclear cells (PMN) with OKT3 monoclonal antibody and,after two days in culture, we analyzed them for expression of LT-α/LT-βcomplex related forms using flow cytofluorometric analysis. In oneexperiment, the results of which are shown in FIG. 1, we cultured freshPBL for 3 days with OKT3 and IL-2 and stained them with a 1:200 dilutionof antisera to native rLT-α0(“LT-4” and “LT-5” panels on FIG. 1, fromrabbits 4 and 5 respectively), denatured rLT-α (“LT-6” panels on FIG. 1,from rabbit 6) and native rTNF (“TNF” panel on FIG. 1, from rabbit 7).We stained cells with postimmune serum (solid lines on FIG. 1 panels) orwith preimmune serum from each animal (dotted lines on FIG. 1 panels).FIG. 1 shows that only anti-rLT-α sera from rabbits 4 and 5 recognizedepitopes on the activated peripheral T cells.

[0170] In the experiment shown in FIG. 2, we treated 11-23.D7 cells withor without 10 ng/ml PMA for 15 hours and stained them as described inthe FIG. 1 experiment, with rabbit 4 anti-rLT-α postimmune serum (solidline on FIG. 2 panels) or with rabbit 4 preimmune serum (dotted line onFIG. 2 panels). As shown in FIG. 2, we found that the T cell hybridomaII-23.D7, which synthesizes LT-α upon phorbol ester (PMA) stimulation,expressed surface LT-related epitopes upon PMA activation.

[0171] To establish that the LT-α-related epitopes on T cells wererelated to LT and not to some contaminant in the CHO-cell derivedrecombinant LT-α preparation, we treated a 1:1000 dilution of antiserumfrom rabbit 4 with PMA-activated, washed II-23.D7 or U937 cells. In theexperiment shown in FIG. 3, we treated a 1 ml sample of anti-rLT-α sera(1:1000 anti-LT-4) with either no cells (- -), 1×10⁸ U937 cells (-O-),1×10⁸ (- -) PMA-activated II-23.D7 cells or 1×10⁷ (- -) PMA-activatedII-23.D7 cells. We added dilutions of absorbed antisera to a limitingamount of rLT-α in a L929 cytotoxicity assay such that a 1:4000 finaldilution was present in the first well. This assay measures the abilityof LT-α to kill a mouse fibroblast cell line, L929, within a 24 hourperiod [L. Green, J. L. Reade, C. F. Ware, “Rapid Colorimetric Assay forCell Viability: Application to the Quantitation of Cytotoxic and GrowthInhibitory Lymphokines,” J. Immunol. Methods, 70:257 (1984)]. After 24hours, we assessed cell viability using a MTT readout. Plotted on FIG. 3is optical density (which is proportional to cell viability) vs. thedilution of absorbed antisera. Data represent the average of duplicatewells and duplicates generally were within the range defined by thesymbol. As shown in FIG. 3, analysis of the neutralizing titer of theabsorbed antisera in the standard L929 cytotoxicity assay demonstratedthat the activated II-23.D7 cells removed the LT-α neutralizingantibodies, whereas U937 cells were ineffective. These data indicatethat the antigenic structures on the membrane surface are actuallyrelated to LT-α.

[0172] We subjected the hybridoma II-23.D7 to a number of furthertreatments to examine a number of trivial explanations for the apparentexistence of LT-α related epitopes on T cell surfaces. First we ruledout the possibility that LT-α:antibody complexes in the antisera couldbind to TNF/LT-α receptors on the hybridoma. Both TNF and LT-α havetrimeric structures which could allow for the presence of antibodybinding epitopes within the complex. However, prior saturation of thecellular TNF receptors with soluble TNF or LT-α had no effect on thesurface staining. Such saturation should have prevented such an immunecomplex from binding to such a receptor.

[0173] A pH 3 lactic acid treatment, which can release bound TNF fromits receptor, had no effect on the signal, suggesting that the LT-α isnot receptor bound. However, experiments utilizing ¹²⁵I-LT-α binding toII-23.D7 cells indicated that receptor bound LT-α was more difficult toremove from its receptor at acidic pH's than TNF.

[0174] Mild trypsinization of the cells prior to staining led to a lossof the signal, indicating that the epitope is a protein. To determinewhether surface-associated LT-α was phosphatidylinositol linked, thecells were treated with a phosphatidylinositol specific phospholipase C.Under conditions where a PI-linked antigen, LFA-3, could be released [A.Peterson et al., “Monoclonal Antibody And Ligand Binding Sites Of The TCell Erythrocyte Receptor (CD2),” Nature, 329:842 (1987)], no effect wasobserved on the LT-α epitope.

[0175] We could not stain CHO cells stably transfected with the LT-αgene, either with or without prior PMA activation, indicating thatantibodies to CHO derived contaminants in the original rLT-α used toimmunize the rabbits were not present in sufficient amounts tocontribute to the staining of II-23.D7 cells. Likewise, antibodiesgenerated against any fetal bovine serum proteins contaminating the LT-αpreparation would be ineffective in staining T cells since the stainingwas performed in 10% fetal calf serum.

[0176] Pretreatment of the anti-rLT-α serum with rLT-α blocked thestaining of LT-α forms on II-23.D7 cells whereas pretreatment with rTNFdid not.

Example 2

[0177] Immunoprecipitation of LT-α-Related Proteins on the T cellHybridoma II-23.D7

[0178] We surface iodinated PMA activated II-23.D7 cells and lysed andsolubilized the cells in detergent. Immunoprecipitation and SDS-PAGEanalysis of the labeled membrane proteins showed that two proteins wererecognized by anti-rLT-α antisera. FIG. 4A shows the results of SDS-PAGEanalysis of the iodinated surface proteins precipitated with eitherpre-immune (PRE) or post-immune (POST) anti-rLT-α serum (from rabbit 4).

[0179] As shown in FIG. 4A, we observed a 25-26 kD molecular weight form(“LT-α”) that correlated with the expected size of LT-α, and we also sawan additional form of approximately 33 kD (“LT-β or p33”). Neither thepreimmune serum from the same rabbit (FIG. 4A column PRE) nor anti-rTNFrabbit serum were able to immunoprecipitate any bands from theiodinated, PMA-activated II-23.D7 cells.

[0180] 1-D partial CNBr peptide mapping of the iodinated bands showedthat the 25-26 kD form was cleaved in a pattern identical to that ofiodinated rLT-α, thus identifying this band as LT-α. In the experimentshown in FIG. 4B, the 25-26 kD and 33 kD bands from panel A wereexcised, subjected to limited CNBr cleavage and electrophoresed on aSDS-PAGE system. For comparison, cleavages of both rTNF and rLT-αperformed in parallel are shown in FIG. 4B. The gels were visualized byautoradiography. Lane 1 represents rTNF, lane 2 represents rLT, lane 3represents LT, and lane 4 represents LT-β. The increased sizes of theCNBr fragments reflect the increased amount of carbohydrate on naturalLT-B. The iodinated 33 kD form was not cleaved by CNBr (lane 4),indicating that it is different from the known LT-α gene product. rTNFwas not cleaved with CNBr (lane 1) due to the absence of methionine inthis protein.

[0181] We undertook metabolic labelling with ³⁵S-methionine and³⁵S-cysteine coupled with immunoprecipitation to characterize furtherthese LT-a-related surface forms. In the case of the TNF/LT-α pair, thedistribution of cysteine and methionine allows one to distinguish bothbetween TNF and LT-α and between forms with and without their signalsequences, as was exploited in studies on the membrane TNF form [M.Kriegler et al., Cell, 53:45-53 (1988)]. In the case of the fullyprocessed cytokines, i.e., secreted forms, TNF contains cysteine and notmethionine, while LT-α contains only methionine and not cysteine. LT-α,however, has two cysteine residues in the signal sequence domain and TNFcontains a methionine residue in this N-terminal region. Separatecultures of II-23.D7 hybridoma cells were labeled with either³⁵S-methionine or ³⁵S-cysteine and immunoreactive proteins wereprecipitated. In an experiment, the results of which are shown in FIG.5, II-23.D7 cells were activated with 10 ng/ml PMA and simultaneouslylabeled for 8 hours with either ³⁵S-methionine or ³⁵S-cysteine. Both themedium and lysed cells were subjected to consecutiveimmunoprecipitations with preimmune (rabbit 4) (P), anti-rTNF (T) andanti-rLT-α (rabbit 4) (L) sera in that order. FIG. 5 shows an SDS-PAGEauto-radiographic analysis of the immunoprecipitates from eithersupernatants containing secreted proteins or the washed cells. “s-TNF”marks the ³⁵S-methionine labeled anti-rTNF immunoprecipitated band fromthe cells that was putatively assigned as the unprocessed 26 kD form ofTNF. “Met” and “Cys” refer to the ³⁵S-labeled amino acid employed. Thoselanes containing ³⁵S-cysteine were exposed for longer periods of timethan the lanes containing ³⁵S-methionine. In the supernatants from thesecells (lanes labeled “secreted”), a 25 kD form of LT (“LT-a”) wasreleased following PMA treatments by those cells that were labeled with³⁵S-methionine, but not by those labeled with ³⁵S-cysteine. This patternis expected for fully processed, secreted LT-a. Longer exposures showedtrace amounts of TNF in the supernatant, and the incorporation of labelwas as expected for fully processed, secreted TNF. We observed theexpression of predominantly LT-α with low levels of TNF also at the mRNAlevel (Shamansky and Ware, unpublished observation). Analysis of thewashed cells (lanes labeled “cellular”) showed that both the 25-26 kDLT-α, along with the 33 kD LT-β, were present. The relative amounts ofthe 25-26 kD and 33 kD forms paralleled those observed using surfaceiodination. The 25-26 kD surface LT-α form lacked cysteine, indicatingprocessing of the leader sequence. The 33 kD form incorporated both³⁵S-methionine and ³⁵S-cysteine. Longer exposures (not shown) of thefilm shown in FIG. 5 revealed the presence of an anti-TNFimmunoprecipitated band from the cells at about 26-27 kD. The bandshowed incorporation of both labeled cysteine and labeled methionine.The labelling was stronger with cysteine. Since the cys:met ratio is 4:1in the 26 kD-TNF form, this labelling pattern confirms the identity ofthis band.

[0182] The presence of LT-β with LT-α in immunoprecipitates from celllysates suggested that either LT-β is antigenically related to LT-α orthat LT-β is bound to LT-α or both. To address this issue 25 kD and 33kD bands from ³⁵S-methionine labeled cells were immunoprecipitated withrabbit polyclonal anti-rLT-α serum, eluted from excised gel slices andsubjected to reimmunoprecipitation with either anti-rLT-α polyclonalserum or mAb. LT-α, but not LT-β, could be immunoprecipitated witheither anti-rLT-α antibodies suggesting that LT-β is not antigenicallyrelated to LT-α. These observations indicated that LT-β is physicallyassociated with LT-α. We believe that the 33 kD protein is unrelatedantigenically to LT-α and simply co-precipitated with LT-α.

[0183] With either surface iodination or metabolic labelling, we wereunable to detect either of the known 55 or 80 kD TNF/LT-α receptor formsassociated with LT-α or TNF. Presumably, this is because the receptorsare rapidly lost during activation of T cells. [C. Ware et al.,“Regulation Of The CTL Lytic Pathway By Tumor Necrosis Factor,” CellularImmunity And The Immunotherapy Of Cancer, UCLA Symposia on Molecular andCell Biology M. T. Lotze and O. J. Finn, Eds. Vol. 135:121-128(Wiley-Liss, Inc. New York) 1990].

Example 3

[0184] Biochemical Characterization of Surface LT-forms on II-23.D7Cells

[0185] We purified the LT-related forms on the surface of PMA-treatedII-23.D7 cells using affinity chromatography. Using immunoprecipitationtechniques, we had noted that both of LT-β and LT-α bound to lentillectin sepharose, indicating a glycoprotein structure. We bounddetergent solubilized PMA-treated II-23.D7 proteins to lentil lectinsepharose and eluted with a-methyl mannoside prior to affinitypurification. We prepared both control IgG and anti-IgG columns to moreaccurately assess those proteins specifically recognized by theanti-rLT-α serum. Low pH elution of the columns led to the release ofabout 100-200 ng of the two LT forms from the anti-rLT-α column.

[0186]FIG. 6A reflects SDS PAGE analysis of the proteins eluted fromanti-rLT-α affinity column prepared from either pre-immune (PRE) orpost-immune (POST) rabbit sera. In FIG. 6B, the 33 kD and 20 kD bandsfrom the gel in panel A were excised and subjected to limiting CNBrcleavage and electrophoresed on a SDS-PAGE system. For comparison, FIG.6B showed CNBr cleavages of rTNF and rLT-α (CHO-derived) performed inparallel. The gels were visualized by silver staining. SDS-PAGE gels ofthe eluate resembled closely gels of immunoprecipitated, surfaceiodinated PMA-treated II-23.D7 cells, indicating that similar proteinshad been purified.

[0187] During the affinity purification, the 25 kD LT-α form appeared tobe cleaved to a 19-20 kD form, i.e., it now co-migrated with the intactrecombinant CHO cell-derived LT-α. The original isolation of naturalLT-α from the RPMI 1788 tumor line also yielded an N-terminally cleaved“des-20” LT-α form. One-dimensional CNBr digests of the affinitypurified proteins showed the cleaved 20 kD LT-α form to have a CNBrcleavage pattern that presumably reflects the truncated nature of thisLT-α form. One of the methionines is lost in the “des-20” LT-α form andhence the cleavage pattern would be different from that of the intactLT-α form. The 33 kD protein (LT-β) generated a doublet upon CNBrcleavage, and from this it was estimated that the single methionine mustlie within 5-20 residues from either the C- or N-terminus. This cleavagepattern shows that the 33 kD protein is significantly different fromknown LT forms. CNBr cleavage analysis of the surface iodinated 33 kDprotein probably gave a similar result; however, the resolutionachievable with iodination was insufficient to visualize the doublet.Staphylococcus V8 digestion of the iodinated rLT-α, rTNF and II-23.D7 LTforms showed the rLT-α and 25-26 kD II-23.D7 LT-α form to be resistantto digestion, confirming the assignment of this protein as LT-α. The 33kD protein was cleaved into several smaller fragments with the patternresembling closely that of rTNF.

[0188] In FIG. 7, immunoprecipitated, surface iodinated proteins wereresolved on SDS-PAGE analysis and the surface-associated 25-26 kDprotein (“sLT-α”) and the 33 kD protein (“LT-β”) bands were excised.Slices were digested with N-glycanase (N-Gly), with a mixture ofneuraminidase and O-glycanase (O-Gly), or with all three enzymes. Thedigested slices were rerun on SDS-PAGE and an autoradiogram of the driedgel is shown. As shown in FIG. 7, immunoprecipitation of iodinatedsurface LT forms followed by digestion with either or both N- andO-glycanases showed the 25-26 kD LT-α form to contain an N-linkedoligosaccharide. The 25-26 kD LT-α form contains only one N-linked sitewhich would correlate well with the size change upon N-glycanasedigestion. Likewise, the 33 kD form (LT-β) lost about 3 kD of size upontreatment with N-glycanase, suggesting the presence of one N-linkedoligosaccharide. In contrast to the 25-26 kD LT-α form, O-glycanasetreatment did not affect the molecular weight of LT-B. The lack ofcleavage by a glycanase, however, is not definitive evidence for thelack of a carbohydrate.

Example 4

[0189] Reimmunoprecipitation of LT-α

[0190] The coprecipitation of LT-β with LT-α suggested that theseproteins are either antigenically related or physically associated. Toaddress this issue we tested whether or not SDS-PAGE separated LT-α(p25) and LT-β proteins could be immunoprecipitated. LT-α and LT-βlabeled with ¹²⁵I or ³⁵S-Met were first partially purified byimmunoprecipitation and separated by SDS-PAGE. The labeled bands wereexcised, rehydrated in buffer, and the proteins eluted. The elutedproteins were then subjected to a second round of immunoprecipitationusing either polyclonal or monoclonal anti-rLT-α antibodies (FIG. 10).Rabbit anti-rLT-α reimmunoprecipitated LT-α (“p25”, lane 2) but not LT-β(lane 3). The anti-rLT-α mAb precipitated LT-α (lane 5) and a 21 kDprotein (“p21”, lane 4), which, as shown below, is a precursor of LT-α;however, it did not precipitate LT-β (lane 6). The results indicate thatafter LT-α and LT-β are separated by SDS-PAGE, both polyclonal andmonoclonal anti-rLT-α antibodies are capable of reacting with LT-α butnot with LT-β. This data provides evidence that LT-β is notantigenically related to LT-α. However, we cannot rule out thepossibility that putative LT-β cross-reactive epitopes are lost afterdenaturation, whereas the LT-α epitopes remain intact.

[0191]FIG. 8 shows the results of reimmuno- precipitation of¹²⁵I-labeled and ³⁵S-Met-labeled p25 and p33 proteins eluted fromSDS-PAGE gels. LT-α and LT-β species from ¹²⁵I-labeled II-23.D7 cells(lanes 2,3) and ³⁵S-labeled cells (lanes 4,5,6) were eluted from gelslices as described above. The elutes were immunoprecipitated witheither the anti-rLT-α serum (lanes 2,3) or the anti-rLT-α mAb (lanes4,5,6), and the reprecipitated proteins analyzed by SDS-PAGE andautoradiography. Lane 1 is a control lane for the identification of LT-αand LT-β.

Example 5

[0192] Isoelectric Focusing of LT-α and LT-β

[0193]FIGS. 9 and 10 (each including an autoradiograph (9A, 10A) and acalibration curve graphing migration distance vs. pH (9B, 10B)) depictisoelectric focusing analysis under denaturing (FIG. 9) and native (FIG.10) conditions. Two-dimensional gel analysis was carried out asdescribed above on ¹²⁵I-labeled LT-α and LT-β that had beenimmunoprecipitated from II-23.D7 cell extracts. The 2-D gel analysis wasperformed under denaturing conditions in the presence of urea (FIG. 9A).In contrast, native IEF was performed in 1% NP-40 without urea.¹²⁵I-labeled II-23.D7 cell extract was first focused on a tube gel at4°. After focusing, the tube gel was cut into 1 cm sections, the focusedproteins eluted from those sections, immunoprecipitated, and analyzed bySDS-PAGE (FIG. 10A). Immunoprecipitated material from gel lanes 1-12correspond to tube gel slices 2-13. pH gradients were generated for boththe denatured and native tube gels based on 1 cm gel increments. Theseare also shown below each autoradiogram as 9B and 10B, respectively.Biochemically, LT-B and LT-α comigrate on a non-denaturing isoelectricfocusing gel, but when the complex is dissociated with urea, the twoproteins run separately. [See FIGS. 9A, 10A] These observations led usto conclude that LT-α and LT-β exist as a complex on the cell surface.

Example 6

[0194] Regulation of LT-α Expression

[0195] Table I set forth below, summarizes the results of a survey usingflow cytofluorometric analysis of various cell types for the expressionof a surface form of LT-α. TABLE I Expression of LT-α and TNF RelatedEpitopes on the Surfaces of Different Cells Surface Expression of: CellTreatment LT-α TNF Peripheral Resting + − Mononuclear cells OKT3 ++ −Leu-4⁺ (CD3) Resting + − OKT3 ++ − PMA − nd. IL-2 ++ nd. Leu-2⁺ (CD8)OKT3 ++ nd. Leu-3⁺ (CD4) OKT3 ++ nd. Leu-M3 Resting − + OKT3/LPS/IFN-γ −++ Leu-19⁺ (NK) IL-2 (LAKs) ++ nd. Leu-16⁺ (B's) Resting +/− − PWM + nd.CTL-clones Control +/− − PMA nd. nd. Allogeneic Stim. ++ nd. Anti-T112 + 3 ++ − T cell Hybridoma Control − − (II-23.D7) PMA ++ − PMA + A23187++ − Hut-78 Control + − PMA + − C8166 Control − nd. PMA + nd RPMI-1788Control − − PMA − − rLT-producing Control − − CHO cell line PMA − −Jurkat +/− nd. HL-60 − − U937 − nd. Raji + nd. K562 +/− nd.

[0196] The most striking observation from these studies was therestriction of surface LT-α expression to T and B cells. Leu-M3, amonocyte marker, and leu-4 (CD3) antibodies were used in two-color flowcytofluorometric analysis to observe each cell population separately.There was an excellent distinction between surface TNF and surface LT-αin this analysis in that monocytes expressed only surface TNF whereas Tcells displayed only surface LT-α. This result is shown in FIG. 11.

[0197] In the experiment depicted in FIG. 11, PBL were treated for 8hours with a mixture of LPS (1 μg/ml), Interferon-γ (200 U/ml) and OKT3(1 ng/ml) and then stained for LT-α (anti-rLT-α serum from rabbit 5) orTNF (anti-rTNF serum from rabbit 7) followed by FITC anti-rabbitlabelling. Cells were counterstained with either phycoerythrin-leu-4, apan T cell marker, or phycoerythrin leu-M3, a monocyte marker. “T cellpanels” were gated for leu-4+cells while the “monocyte” panels weregated for leu-M3+cells. Cells were stained with preimmune (dotted lines)or postimmune sera (solid lines). The monocytic tumor lines HL-60 andU937 did not stain for LT-α. By using two color flow cytofluorometricanalysis, the T4 and T8 subclasses of activated PBL were found todisplay similar levels of surface-associated LT-α. In general, itappears that primary T-cells capable of expressing LT-α are also capableof displaying surface LT-α form(s).

[0198] Examination of three different human donors showed that a surfaceLT-α form was present on freshly isolated, resting peripheral T cells.In the case of PBL, OKT3 activation or simply IL-2 treatment of thecells led to increased expression. By using fluorescence channelnumbers, we have attempted to quantitate both surface LT-α and IL-2receptor (CD25) expression during OKT3 activation. Maximal surface LT-αinduction by OKT3 appeared to precede the peak expression of IL-2receptor (TAC expression) in the bulk culture, thus the surface-bound LTform (LT-α/LT-β complex) appears to be an early T-cell activationantigen. It was found that both anti-T11 and allogeneic antigen werecapable of causing the appearance of LT-α on the surface of clonedcytotoxic T cells. Likewise, PMA stimulation was necessary to induce theappearance of LT-α on the surface of the II-23.D7 hybridoma. It appearsthat T cell activation increases surface LT-α form(s). Peripherallymphocytes, in contrast to the II-23.D7 hybridoma, down-regulatesurface LT-α form(s) very quickly following PMA treatment. Likewise, ina two-color analysis of OKT3 activated PBL populations, Dr⁺ cells, whichshould include T cells in advanced stages of activation, lacked surfaceLT-α form(s).

[0199] Activation of fresh PBL with high levels of IL-2 generatedlymphokine-activated killer cells (LAK cells). As shown in FIG. 12, twocolor flow cytofluorometric analysis using anti-rLT-α and leu-19, aNK/LAK cell marker, showed LAK cell expression of surface LT-α forms toresemble the T cell hybridoma, II-23.D7. In the experiment depicted inFIG. 12, PBL were cultured for 5 days with 20 ng/ml IL-2 and thenstained for a two color analysis with phycoerythrin labeled leu-19 andanti-rLT (rabbit 5) as described above. FIG. 12 shows surface LT-αlevels on leu-19⁺ cells that were stained with preimmune (dotted lines)or postimmune sera (solid lines). Thus, LAK cells appeared to have thehighest levels of surface LT-α forms of any primary cell type.

Example 7

[0200] Functional Relevance of Total TNF or LT-α to T Cell Activation

[0201] To examine the functional relevance of TNF and LT-α to the T cellactivation process, we included the rabbit anti-rLT-α and anti-rTNF serain mixed lymphocyte response (MLR) and OKT3 activation assays. MLR is astandard immunological assay which tests the ability of an individual'sT cells to recognize another person's T cells as foreign and respond totheir presence by proliferating. Table II, set forth below, presentsdata from MLR experiments using various responder/stimulatorcombinations. TABLE II Effects of LT and TNF Antibodies on a 5-Day MixedLymphocyte Culture ³H-Thy. (S.D.) % Change^(a) Cells^(b) Addition cpm ×1000 Responder A none 4.7 (0.8) − Stimulator B* none 4.8 (0.5) − A + B*none 20.3 (3.6)    0% A + B* r-TNF^(c) 28.0 (1.4) +49% A + B* r-LT 32.8(3.2) +80% Responder C none 6.3 (0.8) − Stimulator B* none 4.8 (0.7) −C + B* none 30.0 (5.4)    0% C + B* r-TNF 36.9 (6.0) +28% C + B* r-LT36.1 (5.8) +24% Responder A none 5.3 (0.6) − Stimulator D* none 1.5(0.5) − A + D* none 22.2 (2.6)    0% Responder D none 7.8 (1.0) −Stimulator A* none 1.6 (0.3) − D + A* none 24.3 (7.0)    0% PreimmunePostimmune A + B* Anti-LT-4^(d) 26.8 (2.4) +41%  8.6 (0.2) −74% A + B*Anti-LT-5 27.4 (4.4) +45% 11.0 (1.2) −59% A + B* Anti-LT-6 23.1 (1.0)+18% 25.7 (5.7) +35% A + B* Anti-TNF 26.0 (2.2) +36% 12.6 (2.7) −49% A +B* Anti-TNF mAb* 15.1 (2.2) −33%  4.7 (0.7) −99% C + B* Anti-LT-4^(d)41.1 (3.5) +44% 20.9 (5.8) −36% C + B* Anti-LT-5 35.5 (6.9) +22% 17.8(2.6) −49% C + B* Anti-LT-6 39.4 (7.9) +38% 39.1 (5.3) +36% C + B*Anti-TNF 39.8 (4.3) +39% 24.4 (3.2) −22% C + B* Anti-TNF mAb^(e) 37.8(7.3) +31% 20.6 (1.8) −37% A + D* Anti-LT-5 28.6 (2.1) +37% 12.5 (2.4)−59% A + D* Anti-LT-5 32.8 (6.4) +63% 14.5 (3.6) −47% D + A* Anti-LT-528.0 (1.2) +23% 20.8 (1.4) −21% D + A* Anti-LT-5^(f) 28.1 (2.1) +24%19.2 (0.7) −31%

[0202] As shown in Table II, the neutralizing anti-rLT-α sera (rabbits 4and 5) inhibited the proliferative response as assessed at five dayswhereas preimmune sera or the non-neutralizing anti-rLT-α (rabbit 6)sera had mild stimulatory effects. As previously reported [M. Shalaby etal., J. Immunol., 141:499 (1988)], polyclonal and a monoclonal anti-TNFpreparations were also inhibitory. These assays were carried out underexcess stimulator cell conditions and hence the inhibition may not beoptimized. The serum levels employed in Table II are rather high, but inother experiments (data not shown), antibody dilutions up to 1:1000 werestill inhibitory. PHA or OKT3 stimulated T cell proliferation was alsoinhibited to a lesser extent (data not shown). These data indicated thatLT or LT-related epitopes on T cell surfaces may be involved in T cellactivation.

[0203] A previous study using the MLR assay and neutralizing monoclonalantibodies implicated TNF but not LT-α in T cell activation andsubsequent proliferation in this system. [M. Shalaby et al., J.Immunol., 141:499 (1988)]. In that study, monoclonal anti-rLT-αantibodies had no effect on the MLR assay. Our studies indicate thatneutralizing polyclonal anti-CHO-cell-derived-rLT-α sera were able topartially inhibit the MLR, suggesting a role for some form of LT in thissystem. The reasons for this discrepancy are not clear, although theremay be some differences in the nature of these antibody preparations.The monoclonal antibodies were generated against glutaraldehyde-crosslinked natural LT-α (RPMI 1788 secreted), whereas polyclonal anti-rLT-αsera were prepared using native r-LT-α (recombinant CHO-cell derived)with Freund's adjuvant injected directly into the lymph nodes. The depoteffect in the success of the latter system was probably importantconsidering the difficulties reported in immunizing mice [T. Bringman etal., “Monoclonal Antibodies to Human Tumor Necrosis Factors Alpha andBeta: Application For Affinity Purification, Immunoassays, And AsStructural Probes,” Hybridoma, 6:489 (1987)]. These data suggest thatthe blocking effect of our polyclonal anti-rLT-α sera on the MLR is aresult of recognition of the surface LT form(s) by the sera rather thanrecognition of the conventional soluble LT-α form.

Example 8

[0204] Purification and Initial Sequencing Of LT-α and LT-β

[0205] We obtained N-terminal sequence information for LT-α and LT-β byEdman degradation as described above. We found the sequence of themembrane associated LT-α band to be as follows: Leu Pro Gly Val Gly LeuThr Pro Ser. This sequence matches with the known sequence of secretedLT-α. The Edman degradation analysis revealed that the N-terminalportion of the associated LT-β protein included two possible amino acidsequences: Gly Leu Glu Gly Arg Gly Gln Arg Leu Gln or Gly Leu Glu GlyArg Leu Gln Arg Leu Gln. Subsequent DNA analysis confirmed the formersequence except that glycine was incorrectly identified as glutamine atcycle 7.

Example 9

[0206] Sequencing and Cloning LT-β

[0207] As described above, we obtained sequences for several peptidesusing the method of Abersold et al. Two antisense 17-mer oligonucleotideprobes GTYTCNGGCTCYTCYTC [SEQ ID NO:9] and GTYTCNGGTTCYTCYTC [SEQ ID NO:10] were synthesized to match a portion of the sequence of one of thosepeptides, T-87/T-88. Those probes were radiolabelled with ³²P. Northernanalysis (as described in J. Sambrook et al., Molecular Cloning aLaboratory Manual, 2d ed. (1989)) showed that probe 1368(GTYTCNGGTCYTCYTC) [SEQ ID NO: 10] hybridized strongly to a 0.9-1.1 kbmRNA band that was strongly induced in II-23.D7 cells which had beenpretreated with phorbol ester as previously described. A cDNA library inthe vector pCDM8 was constructed from poly A+mRNA isolated from II-23.D7cells induced with PMA for 6 hours. [See, B. Seed and A. Aruffo, PNAS,84:3365-3369 (1987)]. The library was screened with labelled oligomer1368 and positive clones were isolated following washing with 3 Mtetramethylammonium chloride at 50° C. [See Jacobs et al., Nucleic AcidsResearch, 16, 10:4637-4649 (1988)]. Several clones containing 0.9 kbinserts were subjected to DNA sequence analysis. Clone pCDM8/LT-β-12(clone 12) was found to contain the coding sequence. The other cloneswere identical except for various 1-30 bp truncations at the 5′ end. Onepotential clone (clone 4) contained a frameshift and was used as acontrol in transfection experiments. A termination sequence, AATAAA atposition 862 was found just prior to a poly A tract indicating that theentire 3′ end had been identified. The identified protein sequenceencodes for at least 240 amino acids with a calculated molecular weightof 25,390 and a domain structure typical of a type II membrane protein.The present data suggest that the initiating CTG leads to a processedN-terminus starting with gly 5 (met=1), i.e., the CTG encoded leucine iseither not translated or the leucine-4 residue is further processedyielding the mature N-terminus obtained by amino acid sequencing.

[0208] The 33 kDa size of LT-β results from N-linked glycosylation aspreviously defined and this result is corroborated by the presence ofone potential N-linked carbohydrate site in the amino acid sequence at aposition identical with a similar site found in the CD40 ligand. Atypical N-linked sugar residue can add approximately 3-4 kDa MW, hence,the final molecular weight is close to the observed 30-33 kDa.

[0209] No identical sequences were found within the databases. There isone cysteine residue in the extracellular domain and two methionineswithin the last C-terminal 17 amino acids, in agreement with the verylimited cyanogen bromide cleavage pattern exhibited by this protein.

Example 10

[0210] Expression of LT-β

[0211] The pCDM8/LT-β clone 12 or a control plasmid, clone 4 (pCDM8 witha non-functional cDNA insert), were introduced by electroporation intoCHO dhfr- and a CHO cell stably transfected with human LT-α. After threedays, cells were removed with Ca/Mg-free Hank's solution with 5 mM EDTAand stained for FACS analysis as described above using either 10 μg/nlcontrol IgGI or anti-LT monoclonal antibody (Boehringer-Mannheim)followed by labelling of bound immunoglobulin with either a FITC orphycoerythrin labelled goat anti-mouse preparation.

[0212] In a different experiment, COS cells were electroporated witheither clone 4 or clone 12 LT-β cDNA in pCDM8 in the presence or absenceof an equal amount of human LT-α cDNA also in the pCDM8 vector andstained for FACS analysis after three days as above. Only COS cellsexpressing LT-α displayed surface lymphotoxin upon transfection with afunctional LT-β DNA, i.e., clone 12.

[0213] Clone 12 lacks an initiating ATG codon, but does possess severalCTG initiating codons and hence this expression experiment shows thatone or several of the 5′ CTG codons can initiate translation. CTG codonsare known to serve as initiating sites for translation in severaleukaryotic proteins. [M. Kozak, J. Cell. Biol., 115:4 (1991)]. Similarresults were observed using the dual transfection system such that onlyCOS cells receiving both LT-α and LT-β DNA displayed substantial surfaceLT-α in a FACS analysis.

[0214] Northern analysis of II-23.D7 cells showed hybridization of theLT-β cDNA to a 0.9-1.0 kb mRNA indicating that the cloned cDNArepresents essentially all of the transcribed gene. See FIG. 15. TheLT-β gene was expressed at low levels in untreated II-23.D7 hybridomacells; however, upon cell activation with phorbol ester mRNA levelsincreased dramatically. See FIG. 16.

Example 11

[0215] Homology Between LT-β and Other Members of the TNF Family OfLymphokines

[0216] Cloning of the cDNA encoding LT-β revealed that LT-β is a type-IImembrane protein with significant homology to TNF, LT-α and the ligandfor the CD40 receptor. These proteins are known to bind to members ofthe TNF/NGF receptor family. LT-β, TNF, LT-α and the ligand for theCD-40 receptor share four regions of sequence conservation in theextracellular domain. See FIG. 13. These domains are located on the faceof the TNF and LT-α crystal structures and are likely to be involved inintersubunit interactions.

Example 12

[0217] Determination of the 5′ End of LT-β Reveals Several PossibleStart Sites

[0218] The 5′ mRNA sequence was determined by primer extension analysis.Primer extension analysis revealed that the transcriptional start sitefor the LT-β protein was approximately 7-9 base pairs upstream of themethionine ATG. Thus, the mRNA possesses at least 3 possible translationstart sites, the Met-1, Leu-4 and Leu-6 codons. Transient experimentswith clone 12 showed that one or both of the Leu-4 or Leu-6 start sitesis functional. The 5′ mRNA sequence was verified by determining the LT-βgenomic sequence using a cosmid clone 031A, described above, anddideoxynucleic acid sequencing.

1 23 1 726 DNA Homo Sapien 1 ctggggctgg agggcagggg tgggaggctc caggggaggggttccctcct gctagctgtg 60 gcaggagcca cttctctggt gaccttgttg ctggcggtgcctatcactgt cctggctgtg 120 ctggccttag tgccccagga tcagggagga ctggtaacggagacggccga ccccggggca 180 caggcccagc aaggactggg gtttcagaag ctgccagaggaggagccaga aacagatctc 240 agccccgggc tcccagctgc ccacctcata ggcgctccgctgaaggggca ggggctaggc 300 tgggagacga cgaaggaaca ggcgtttctg acgagcgggacgcagttctc ggacgccgag 360 gggctggcgc tcccgcagga cggcctctat tacctctactgtctcgtcgg ctaccggggc 420 cgggcgcccc ctggcggcgg ggacccccag ggccgctcggtcacgctgcg cagctctctg 480 taccgggcgg ggggcgccta cgggccgggc actcccgagctgctgctcga gggcgccgag 540 acggtgactc cagtgctgga cccggccagg agacaagggtacgggcctct ctggtacacg 600 agcgtggggt tcggcggcct ggtgcagctc cggaggggcgagagggtgta cgtcaacatc 660 agtcaccccg atatggtgga cttcgcgaga gggaagaccttctttggggc cgtgatggtg 720 gggtga 726 2 241 PRT Homo Sapien 2 Leu Gly LeuGlu Gly Arg Gly Gly Arg Leu Gln Gly Arg Gly Ser Leu 1 5 10 15 Leu LeuAla Val Ala Gly Ala Thr Ser Leu Val Thr Leu Leu Leu Ala 20 25 30 Val ProIle Thr Val Leu Ala Val Leu Ala Leu Val Pro Gln Asp Gln 35 40 45 Gly GlyLeu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln Ala Gln Gln 50 55 60 Gly LeuGly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu Thr Asp Leu 65 70 75 80 SerPro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro Leu Lys Gly 85 90 95 GlnGly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe Leu Thr Ser 100 105 110Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro Gln Asp Gly 115 120125 Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg Ala Pro Pro 130135 140 Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg Ser Ser Leu145 150 155 160 Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu LeuLeu Leu 165 170 175 Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro AlaArg Arg Gln 180 185 190 Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly PheGly Gly Leu Val 195 200 205 Gln Leu Arg Arg Gly Glu Arg Val Tyr Val AsnIle Ser His Pro Asp 210 215 220 Met Val Asp Phe Ala Arg Gly Lys Thr PhePhe Gly Ala Val Met Val 225 230 235 240 Gly 3 606 DNA Homo Sapien 3ctggccttag tgccccagga tcagggagga ctggtaacgg agacggccga ccccggggca 60caggcccagc aaggactggg gtttcagaag ctgccagagg aggagccaga aacagatctc 120agccccgggc tcccagctgc ccacctcata ggcgctccgc tgaaggggca ggggctaggc 180tgggagacga cgaaggaaca ggcgtttctg acgagcggga cgcagttctc ggacgccgag 240gggctggcgc tcccgcagga cggcctctat tacctctact gtctcgtcgg ctaccggggc 300cgggcgcccc ctggcggcgg ggacccccag ggccgctcgg tcacgctgcg cagctctctg 360taccgggcgg ggggcgccta cgggccgggc actcccgagc tgctgctcga gggcgccgag 420acggtgactc cagtgctgga cccggccagg agacaagggt acgggcctct ctggtacacg 480agcgtggggt tcggcggcct ggtgcagctc cggaggggcg agagggtgta cgtcaacatc 540agtcaccccg atatggtgga cttcgcgaga gggaagacct tctttggggc cgtgatggtg 600gggtga 606 4 201 PRT Homo Sapien 4 Leu Ala Leu Val Pro Gln Asp Gln GlyGly Leu Val Thr Glu Thr Ala 1 5 10 15 Asp Pro Gly Ala Gln Ala Gln GlnGly Leu Gly Phe Gln Lys Leu Pro 20 25 30 Glu Glu Glu Pro Glu Thr Asp LeuSer Pro Gly Leu Pro Ala Ala His 35 40 45 Leu Ile Gly Ala Pro Leu Lys GlyGln Gly Leu Gly Trp Glu Thr Thr 50 55 60 Lys Glu Gln Ala Phe Leu Thr SerGly Thr Gln Phe Ser Asp Ala Glu 65 70 75 80 Gly Leu Ala Leu Pro Gln AspGly Leu Tyr Tyr Leu Tyr Cys Leu Val 85 90 95 Gly Tyr Arg Gly Arg Ala ProPro Gly Gly Gly Asp Pro Gln Gly Arg 100 105 110 Ser Val Thr Leu Arg SerSer Leu Tyr Arg Ala Gly Gly Ala Tyr Gly 115 120 125 Pro Gly Thr Pro GluLeu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro 130 135 140 Val Leu Asp ProAla Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr 145 150 155 160 Ser ValGly Phe Gly Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val 165 170 175 TyrVal Asn Ile Ser His Pro Asp Met Val Asp Phe Ala Arg Gly Lys 180 185 190Thr Phe Phe Gly Ala Val Met Val Gly 195 200 5 450 DNA Homo Sapien 5ccgctgaagg ggcaggggct aggctgggag acgacgaagg aacaggcgtt tctgacgagc 60gggacgcagt tctcggacgc cgaggggctg gcgctcccgc aggacggcct ctattacctc 120tactgtctcg tcggctaccg gggccgggcg ccccctggcg gcggggaccc ccagggccgc 180tcggtcacgc tgcgcagctc tctgtaccgg gcggggggcg cctacgggcc gggcactccc 240gagctgctgc tcgagggcgc cgagacggtg actccagtgc tggacccggc caggagacaa 300gggtacgggc ctctctggta cacgagcgtg gggttcggcg gcctggtgca gctccggagg 360ggcgagaggg tgtacgtcaa catcagtcac cccgatatgg tggacttcgc gagagggaag 420accttctttg gggccgtgat ggtggggtga 450 6 149 PRT Homo Sapien 6 Pro Leu LysGly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala 1 5 10 15 Phe LeuThr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu 20 25 30 Pro GlnAsp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly 35 40 45 Arg AlaPro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu 50 55 60 Arg SerSer Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro 65 70 75 80 GluLeu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro 85 90 95 AlaArg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe 100 105 110Gly Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile 115 120125 Ser His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly 130135 140 Ala Val Met Val Gly 145 7 156 DNA Homo Sapien 7 ctggccttagtgccccagga tcagggagga ctggtaacgg agacggccga ccccggggca 60 caggcccagcaaggactggg gtttcagaag ctgccagagg aggagccaga aacagatctc 120 agccccgggctcccagctgc ccacctcata ggcgct 156 8 52 PRT Homo Sapien 8 Leu Ala Leu ValPro Gln Asp Gln Gly Gly Leu Val Thr Glu Thr Ala 1 5 10 15 Asp Pro GlyAla Gln Ala Gln Gln Gly Leu Gly Phe Gln Lys Leu Pro 20 25 30 Glu Glu GluPro Glu Thr Asp Leu Ser Pro Gly Leu Pro Ala Ala His 35 40 45 Leu Ile GlyAla 50 9 17 DNA Homo Sapien misc_feature 6 n = A,T,C or G 9 gtytcnggctcytcytc 17 10 17 DNA Homo Sapien misc_feature 6 n = A,T,C or G 10gtytcnggtt cytcytc 17 11 18 DNA Homo Sapien 11 atgggggcac tggggctg 18 1219 DNA Homo Sapien 12 gcggccgctt tagagcaca 19 13 27 DNA Homo Sapien 13gacagtgata ggcaccgcca gcaacaa 27 14 34 PRT Homo Sapien VARIANT 7, 30, 31Xaa = Any Amino Acid 14 Gly Leu Glu Gly Arg Gly Xaa Arg Leu Gln Gly ArgGly Ser Leu Leu 1 5 10 15 Leu Ala Val Ala Gly Ala Thr Gly Leu Val ThrLeu Leu Xaa Xaa Val 20 25 30 Pro Ile 15 25 PRT Homo Sapien 15 Leu ProGlu Glu Glu Pro Glu Thr Asp Leu Ser Pro Gly Leu Pro Ala 1 5 10 15 AlaHis Leu Ile Gly Ala Pro Leu Lys 20 25 16 25 PRT Homo Sapien VARIANT 1Xaa = Any Amino Acid 16 Xaa Gln Ala Phe Leu Thr Ser Gly Thr Gln Phe SerAsp Ala Glu Gly 1 5 10 15 Leu Ala Leu Pro Gln Asp Gly Leu Tyr 20 25 17 8PRT Homo Sapien VARIANT 1, 5, 6 Xaa = Any Amino Acid 17 Xaa Gln Gly LeuXaa Xaa Glu Thr 1 5 18 5 PRT Homo Sapien 18 Ser Ser Leu Tyr Arg 1 5 1926 PRT Homo Sapien 19 Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu LeuLeu Leu Leu Glu 1 5 10 15 Gly Ala Glu Thr Val Thr Pro Val Leu Asp 20 2520 233 PRT Homo Sapien 20 Met Ser Thr Glu Ser Met Ile Arg Asp Val GluLeu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln GlySer Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val AlaGly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly ProGln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu AlaGln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro ValAla His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp LeuAsn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu ArgAsp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile TyrSer Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His ValLeu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr GlnThr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln ArgGlu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro IleTyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 SerAla Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 21 260 PRT Murinae gen. sp.21 Met Ile Glu Thr Tyr Ser Gln Pro Ser Pro Arg Ser Val Ala Thr Gly 1 510 15 Leu Pro Ala Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 2025 30 Ile Thr Gln Met Ile Gly Ser Val Leu Phe Ala Val Tyr Leu His Arg 3540 45 Arg Leu Asp Lys Val Glu Glu Glu Val Asn Leu His Glu Asp Phe Val 5055 60 Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys Gly Glu Gly Ser Leu Ser 6570 75 80 Leu Leu Asn Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu Val Lys85 90 95 Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met100 105 110 Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val SerGlu 115 120 125 Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys LysGly Tyr 130 135 140 Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn GlyLys Gln Leu 145 150 155 160 Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val TyrThr Gln Val Thr Phe 165 170 175 Cys Ser Asn Arg Glu Pro Ser Ser Gln ArgPro Phe Ile Val Gly Leu 180 185 190 Trp Leu Lys Pro Ser Ile Gly Ser GluArg Ile Leu Leu Lys Ala Ala 195 200 205 Asn Thr His Ser Ser Ser Gln LeuCys Glu Gln Gln Ser Val His Leu 210 215 220 Gly Gly Val Phe Glu Leu GlnAla Gly Ala Ser Val Phe Val Asn Val 225 230 235 240 Thr Glu Ala Ser GlnVal Ile His Arg Val Gly Phe Ser Ser Phe Gly 245 250 255 Leu Leu Lys Leu260 22 240 PRT Homo Sapien 22 Gly Leu Glu Gly Arg Gly Gly Arg Leu GlnGly Arg Gly Ser Leu Leu 1 5 10 15 Leu Ala Val Ala Gly Ala Thr Ser LeuVal Thr Leu Leu Leu Ala Val 20 25 30 Pro Ile Thr Val Leu Ala Val Leu AlaLeu Val Pro Gln Asp Gln Gly 35 40 45 Gly Leu Val Thr Glu Thr Ala Asp ProGly Ala Gln Ala Gln Gln Gly 50 55 60 Leu Gly Phe Gln Lys Leu Pro Glu GluGlu Pro Glu Thr Asp Leu Ser 65 70 75 80 Pro Gly Leu Pro Ala Ala His LeuIle Gly Ala Pro Leu Lys Gly Gln 85 90 95 Gly Leu Gly Trp Glu Thr Thr LysGlu Gln Ala Phe Leu Thr Ser Gly 100 105 110 Thr Gln Phe Ser Asp Ala GluGly Leu Ala Leu Pro Gln Asp Gly Leu 115 120 125 Tyr Tyr Leu Tyr Cys LeuVal Gly Tyr Arg Gly Arg Ala Pro Pro Gly 130 135 140 Gly Gly Asp Pro GlnGly Arg Ser Val Thr Leu Arg Ser Ser Leu Tyr 145 150 155 160 Arg Ala GlyGly Ala Tyr Gly Pro Gly Thr Pro Glu Leu Leu Leu Glu 165 170 175 Gly AlaGlu Thr Val Thr Pro Val Leu Asp Pro Ala Arg Arg Gln Gly 180 185 190 TyrGly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly Gly Leu Val Gln 195 200 205Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser His Pro Asp Met 210 215220 Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala Val Met Val Gly 225230 235 240 23 205 PRT Homo Sapien 23 Met Thr Pro Pro Glu Arg Leu PheLeu Pro Arg Val Cys Gly Thr Thr 1 5 10 15 Leu His Leu Leu Leu Leu GlyLeu Leu Leu Val Leu Leu Pro Gly Ala 20 25 30 Gln Gly Leu Pro Gly Val GlyLeu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45 Arg Gln His Pro Lys Met HisLeu Ala His Ser Thr Leu Lys Pro Ala 50 55 60 Ala His Leu Ile Gly Asp ProSer Lys Gln Asn Ser Leu Leu Trp Arg 65 70 75 80 Ala Asn Thr Asp Arg AlaPhe Leu Gln Asp Gly Phe Ser Leu Ser Asn 85 90 95 Asn Ser Leu Leu Val ProThr Ser Gly Ile Tyr Phe Val Tyr Ser Gln 100 105 110 Val Val Phe Ser GlyLys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro 115 120 125 Leu Tyr Leu AlaHis Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe 130 135 140 His Val ProLeu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln 145 150 155 160 GluPro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr 165 170 175Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val 180 185190 Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu 195 200 205

We claim:
 1. Lymphotoxin-β, a lymphocyte membrane-type polypeptidecomprising SEQ ID NO:2.
 2. The polypeptide according to claim 1 whereinthe polypeptide is associated with a cell surface.
 3. The polypeptideaccording to claim 2 wherein the polypeptide is associated with thesurface of OKT3-stimulated primary T cells, antigen-specific IL-2dependent CTL clones, and a PMA-stimulated human T cell hybridoma,II-23.D7.
 4. A soluble lymphotoxin-13 peptide comprising an amino acidsequence selected from the group consisting of: (a) SEQ ID NO:4; (b) SEQID NO:6; and (c) an amino acid sequence represented by the followingformula: X—SEQ ID NO:6, wherein X comprises one or more of the aminoacid residues starting from the 3′ end of SEQ ID NO:8.
 5. A peptideaccording to claim 4 further comprising a leader sequence at the 5′ end.6. A polypeptide comprising an amino acid sequence that is encoded by aDNA from the group consisting of: (a) a DNA sequence comprising SEQ IDNO: 1; (b) DNA sequences that hybridize to the DNA defined by SEQ IDNO:1 and that code on expression for a polypeptide that is substantiallyhomologous with lymphotoxin-β; and (c) DNA comprising degeneratenucleotide sequences that code for the polypeptide that is encoded bythe DNA sequence defined by SEQ ID NO:1.
 7. A polypeptide comprising anamino acid sequence that is encoded by a DNA from the group consistingof: (a) a DNA sequence comprising SEQ ID NO:3; (b) a DNA sequencecomprising SEQ ID NO:5; (c) a DNA sequence represented by the followingformula: X—SEQ ID NO:5, wherein X comprises one or more of thenucleoside triplets starting from the 3′ end of SEQ ID NO:7; (d) DNAsequences that hybridize to any one of SEQ ID NO:3, SEQ ID NO:5 and thesequence according to part (c) above and that code on expression for apolypeptide that is substantially homologous with a solublelymphotoxin-β peptide; and (e) a DNA sequence comprising degeneratenucleotide sequences that code for the polypeptide encoded for any oneof SEQ ID NO:3, SEQ ID NO:5 and the sequence according to part (c)above.
 8. An engineered polypeptide comprising the amino acid sequencedefined by SEQ ID NO:2 wherein the sequence Leu Gly Leu is cleaved fromthe 5′ end of said sequence and replaced by a single Met or Leu residue.9. An isolated DNA sequence selected from the group consisting of: (a) aDNA sequence comprising the nucleotide sequence defined by SEQ ID NO: 1;(b) a DNA sequence that hybridizes with the DNA sequence defined by SEQID NO:1 and that codes on expression for a polypeptide that issubstantially homologous with lymphotoxin-β; and (c) a DNA sequencecomprising degenerate nucleotide sequences that code for lymphotoxin-β.10. An isolated DNA sequence selected from the group consisting of: (a)a DNA sequence comprising the nucleotide sequence defined by SEQ IDNO:3; (b) a DNA sequence comprising the nucleotide sequence defined bySEQ ID NO:5; (c) a DNA sequence comprising the nucleotide sequenceaccording to claim 7(c); (d) DNA sequences that hybridize to a DNAsequence as defined by any one of SEQ ID NO:3, SEQ ID NO:5 or thesequence according to claim 7(c) and that code on expression for apolypeptide that is substantially homologous with a solublelymphotoxin-β peptide; and (e) a DNA sequence comprising degeneratenucleotide sequences that code for a soluble lymphotoxin-β peptide. 11.An engineered DNA sequence comprising the nucleotide sequence defined bySEQ ID NO:1 wherein the nucleotides CTGGGGCTG are cleaved from the 5′end of said sequence and replaced by a single start codon.
 12. Arecombinant DNA molecule comprising a DNA sequence selected from thegroup consisting of: (a) a DNA sequence defined by SEQ ID NO:1; (b) aDNA sequence defined by SEQ ID NO:3; (c) a DNA sequence defined by SEQID NO:5; (d) a DNA sequence according to claim 7(c); (e) a DNA sequenceaccording to claim 11; (f) a DNA sequence that hybridizes with the DNAsequences defined by any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5and the sequence according to claim 7(c) and that codes on expressionfor lymphotoxin-β or a soluble lymphotoxin-β peptide; (g) a DNA sequencecomprising degenerate nucleotide sequences that codes for lymphotoxin-β;and (h) a DNA sequence comprising degenerate nucleotide sequences thatcodes for a soluble lymphotoxin-β peptide.
 13. A host selected from thegroup consisting of unicellular hosts, animal cells in culture and humancells in culture, transfected with the recombinant DNA molecule of claim12.
 14. The host according to claim 13 selected from the group of tumorinfiltrating lymphocytes, lymphokine activated killer cells, killercells and genetically engineered tumor cells removed from a patient. 15.A method for producing the polypeptide of any one of claims 1 to 8, saidmethod comprising the steps of culturing a transformed host according toclaim 13 and collecting the polypeptide.
 16. A polypeptide complexcomprising a first polypeptide selected from a group consisting of theamino acid sequence defined by any one of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, a polypeptide according to claim 8, and soluble lymphotoxin-βpeptide according to claim 4(c), and a second polypeptide selected fromthe group consisting of lymphotoxin-α, native human or animallymphotoxin, recombinant lymphotoxin, soluble lymphotoxin, secretedlymphotoxin, or lymphotoxin or lymphotoxin-active fragments of any ofthe above.
 17. A polypeptide complex comprising a plurality oflymphotoxin-β polypeptide units.
 18. A polypeptide complex according toclaim 16 wherein the complex is associated with a cell surface.
 19. Apolypeptide complex according to claim 18 wherein the first polypeptideis associated with the surface of OKT3-stimulated primary T cells,antigen-specific IL-2 dependent CTL clones, and a PMA-stimulatednon-lymphotoxin human T cell hybridoma, II-23.D7.
 20. A method forproducing lymphotoxin epitopes on the surface of a cell comprising thesteps of transfecting the cell with a recombinant DNA molecule accordingto claim 12 and expressing that DNA in the cell.
 21. A method forenhancing the targeting tumorcidal activity of tumor infiltratinglymphocytes comprising the steps of transfecting the lymphocytes with arecombinant DNA molecule according to claim 12 and introducing thetransformed lymphocytes to a patient.
 22. The method according to claim21, wherein the transformed lymphocytes are incubated with a lymphokinebefore or after transfection with the recombinant DNA molecule accordingto claim
 12. 23. The method according to claim 22, wherein thelymphokine is IL-2.
 24. A composition for preventing, treating orlessening the advancement, severity or effects of HIV infection,neoplasia, inflammation or inflammatory disease, or autoimmune diseasecomprising an effective amount of a polypeptide selected from the groupconsisting of a polypeptide according to any one of claims 1 to 8, apolypeptide complex according to any one of claims 16-19, antibodies toany one of the above, or a combination of any of the above, and apharmaceutically acceptable carrier.
 25. A method for preventing,treating or lessening the advancement, severity or effects of HIVinfection, neoplasia, inflammation or inflammatory diseases, orautoimmune disease comprising administering an effective amount of apolypeptide selected from the group consisting of a polypeptideaccording to any one of claims 1-8, a polypeptide complex according toany one of claims 16-19, antibodies to any one of the above, or acombination of any of the above, and a pharmaceutically acceptablecarrier.
 26. A composition for suppressing the immune system comprisingan effective amount of a polypeptide selected from the group consistingof a polypeptide according to any one of claims 1 to 8, a polypeptidecomplex according to any one of claims 16-19, antibodies to any one ofthe above, or a combination of any of the above, and a pharmaceuticallyacceptable carrier.
 27. A method for suppressing the immune systemcomprising administering an effective amount of a polypeptide selectedfrom the group consisting of a polypeptide according to any one ofclaims 1-8, a polypeptide complex according to any one of claims 16-19,antibodies to any one of the above, or a combination of any of theabove, and a pharmaceutically acceptable carrier.
 28. A nucleotidesequence coding for lymphotoxin-β comprising the nucleotide sequencerepresented by SEQ ID NO:1 and further comprising an engineerednucleotide sequence at the 5′ end wherein said engineered sequencecomprises a functional start codon that is either ATG or CTG and whereinany other codon within said engineered sequence coding for leucine isnot CTG.